Matrix metalloproteinase inhibitors

ABSTRACT

Thus the present invention describes diagnostic agents comprising a diagnostic metal and a compound, wherein the compound comprises: 1-10 targeting moieties; a chelator; and 0-1 linking groups between the targeting moiety and chelator; wherein the targeting moiety is a matrix metalloproteinase inhibitor; and wherein the chelator is capable of conjugating to the diagnostic metal. The present invention also provides novel compositions of the compounds of the invention, kits, and their uses in diagnosis of diseases associated with MMPs.

FIELD OF THE INVENTION

The present invention provides novel compounds useful for the diagnosisof cardiovascular pathologies associated with extracellular matrixdegradation, such as atherosclerosis, heart failure, and restenosis,methods of imaging these pathologies in a patient, and pharmaceuticalcompositions comprising the compounds. The pharmaceuticals are comprisedof a targeting moiety that inhibits a matrix metalloproteinase that isexpressed in these pathologies, an optional linking group, and adiagnostically effective imageable moiety. The imageable moiety is agamma ray or positron emitting radioisotope, a magnetic resonanceimaging contrast agent, an X-ray contrast agent, or an ultrasoundcontrast agent.

BACKGROUND OF THE INVENTION

The ability to detect increased levels of matrix metalloproteinases(MMPs) in the heart would be extremely useful for the detection oftissue degradation which occurs in many heart conditions. Thecomposition and vulnerability of atheromatous plaque in the coronaryarteries has recently been recognized as a key determinant inthrombus-mediated acute coronary events, such as unstable angina,myocardial infarction and death (Falk E, Shah P K and Fuster V;Circulation 1995; 92: 657-671). Among the many components involved inthe inflammatory atheromatous plaque are macrophages which secrete thematrix metalloproteinases (Davies M J; Circulation 1996; 94: 2013-2020).The MMPs are a family of enzymes which specialize in the cleavage of theusually protease-resistant fibrillar extracellular matrix components ofthe heart, such as collagen. These extracellular matrix proteins conferstrength to the fibrous cap of atheroma (Libby P, Circulation 1995; 91:2844-2850).

Macrophages which accumulate in areas of inflammation such asatherosclerotic plaques release these MMPs which degrade connectivetissue matrix proteins (Falk, 1995). In fact, studies have demonstratedthat both the metalloproteinases and their mRNA are present inatherosclerotic plaques (Coker M L, Thomas C V, Clair M J, et al.; Am.J. Physiol. 1998; 274:H1516-1523; Dollery C M, McEwan J R, Henney A., etal.; Circ. Res. 1995; 77:863-868; Henney A., Wakeley P., Davies M., etal.; Proc Natl Acad Sci 1991; 88:8154-8158), particularly in thevulnerable regions of human atherosclerotic plaques (Galis Z, Sukhova G,Lark M. and Libby P.; J Clin Invest. 1994; 94: 2493-2503). Amongst themetalloproteinases that may be released by macrophages present at thesite of human atheroma are interstitial collagenase (MMP-1), gelatinasesA and B (MMP-2 and MMP-9, respectively) and stromelysin (MMP-3 Moreno PR, Falk E., Palacious I F et al.; Circulation 1994; 90: 775-778).Although all MMPs may be elevated at the site of human atheroma, it hasbeen suggested that gelatinase B may be one of the most prevalent MMPsin the plaque because it can be expressed by virtually all activatedmacrophages (Brown D., Hibbs M, Kerney M., et al.; Circulation 1995; 91:2125-2131). The MMP-9 has also been shown to be more prevalent inatherectomy material from unstable angina relative to stable anginapatients (Brown, 1995).

The left ventricular extracellular matrix, containing a variety ofcollagens and elastin, are also proposed to participate in themaintenance of left ventricle (LV) geometry. Therefore, alterations inthese extracellular components of the myocardium may influence LVfunction and be a marker of progressive changes associated with LVdegeneration and ultimately heart failure (Coker, 1998).

In the situation of congestive heart failure (CHF), the relationship ofCHF state to MMP activity in the LV remains somewhat unclear, at leastin the clinical setting. In pre-clinical models of CHF, however, thefunctional changes in the LV have been correlated with increased MMPactivity. For example, in a pig model of CHF, the decrease in LVfunction was observed to coincide with a marked increase in MMP-1(˜300%), MMP-2 (˜200%), and MMP-3 (500%) (Coker, 1998). Moderateischemia and reperfusion in a pig model has been demonstrated toselectively activate MMP-9 (Lu L, et al., Circulation, 1999, 100 Suppl.1, I-12). Similarly in a dog model of CHF the levels of gelatinases(e.g. MMP-2 and MMP-9) were found to be elevated in severe heart failure(Armstrong P W, Moe G W, et al., Can J Cardiol 1994; 10: 214-220). Thelevels of MMP-2 and MT1-MMP (membrane type MMP, MMP-14) were found to beincreased in biopsy samples of human myocytes from patients sufferingfrom dilated cardiomyopathy (Bond B R, et al., Circulation, 1999, 100Suppl. 1, I-12).

Ahrens, et al. U.S. Pat. No. 5,674,754 discloses methods for thedetection of Matrix Metallo-Proteinase No. 9, using antibodies whichselectively recognize pro-MMP-9 and complexes of pro-MMP-9 with tissueinhibitor of matrix metallo proteinase-1 (TIMP-1), with no substantialbinding to active MMP-9. Venkatesan, et al. U.S. Pat. No. 6,172,057discloses non-peptide inhibitors of matrix metalloproteinases (MMPs) andTNF-.alpha. converting enzyme (TACE) for the treatment of arthritis,tumor metastasis, tissue ulceration, abnormal wound healing, periodontaldisease, bone disease, diabetes (insulin resistance) and HIV infection.

Pathologically, MMPs have been identified as associated with severaldisease states. For example, anomalous MMP-2 levels have been detectedin lung cancer patients, where it was observed that serum MMP-2 levelswere significantly elevated in stage 1V disease and in those patientswith distant metastases as compared to normal sera values (Garbisa etal., 1992, Cancer Res., 53: 4548, incorporated herein by reference.).Also, it was observed that plasma levels of MMP-9 were elevated inpatients with colon and breast cancer (Zucker et al., 1993, Cancer Res.53: 140 incorporated herein by reference).

Elevated levels of stromelysin (MMP-3) and interstitial collagenase(MMP-1) have been noted in synovial fluid derived from rheumatoidarthritis patients as compared to post-traumatic knee injury (Walakovitset al., 1992, Arth. Rheum., 35: 35) incorporated herein by reference.Increased levels of mRNA expression for collagenase type I (MMP-1) andcollagenase type IV (MMP-2) have been shown to be increased inulcerative colitis as compared to Crohn's disease and controls (Mattheset al., 1992, Gastroenterology, Abstract 661, incorporated herein byreference). Furthrmore, Anthony et al., 1992, Gastroenterology, Abstract591, demonstrated increased immuno-histochemical expression of thegelatinase antigen in a rabbit model of chronic inflammatory colitis.

It has been shown that the gelatinase MMPs are most intimately involvedwith the growth and spread of tumors. It is known that the level ofexpression of gelatinase is elevated in malignancies, and thatgelatinase can degrade the basement membrane which leads to tumormetastasis. Angiogenesis, required for the growth of solid tumors, hasalso recently been shown to have a gelatinase component to itspathology. Furthermore, there is evidence to suggest that gelatinase isinvolved in plaque rupture associated with atherosclerosis. Otherconditions mediated by MMPs are restenosis, MMP-mediated osteopenias,inflammatory diseases of the central nervous system, skin aging, tumorgrowth, osteoarthritis, rheumatoid arthritis, septic arthritis, cornealulceration, abnormal wound healing, bone disease, proteinuria,aneurysmal aortic disease, degenerative cartilage loss followingtraumatic joint injury, demyelinating diseases of the nervous system,cirrhosis of the liver, glomerular disease of the kidney, prematurerupture of fetal membranes, inflammatory bowel disease, periodontaldisease, age related macular degeneration, diabetic retinopathy,proliferative vitreoretinopathy, retinopathy of prematurity, ocularinflammation, keratoconus, Sjogren's syndrome, myopia, ocular tumors,ocular angiogenesis/neo-vascularization and corneal graft rejection. Forrecent reviews, see: (1) Recent Advances in Matrix MetalloproteinaseInhibitor Research, R. P. Beckett, A. H. Davidson, A. H. Drummond, P.Huxley and M. Whittaker, Research Focus, Vol. 1, 16-26, (1996), (2)Curr. Opin. Ther. Patents (1994) 4(1): 7-16, (3) Curr. Medicinal Chem.(1995) 2: 743-762, (4) Exp. Opin. Ther. Patents (1995) 5(2): 1087-110,(5) Exp. Opin. Ther. Patents (1995) 5(12): 1287-1196, all of which areincorporated herein by reference.

Therefore, the present imaging agents targeted to one or more MMP'swould be very useful for detecting and monitoring the degree ofextracellular matrix degradation in CHF, atherosclerosis and otherdegradative disease processes. These imaging agents, containing a liganddirected at one or more MMP's (e.g. MMP-1, MMP-2, MMP-3, MMP-9), willlocalize a diagnostic imaging probe to the site of pathology for thepurpose of non-invasive imaging of these diseases. The imaging agent maybe a MMP inhibitor linked to radioisotopes which are known to be usefulfor imaging by gamma scintigraphy or positron emission tomography (PET).Alternatively, the MMP targeting ligand could be bound to a single ormultiple chelator moieties for attachment of one or more paramagneticmetal atoms, which would cause a local change in magnetic properties,such as relaxivity or susceptibility, at the site of tissue damage,which could then be imaged with magnetic resonance imaging systems.Alternatively, the MMP inhibitor can be bound to a phospholipid orpolymer material which would be used to encapsulate/stabilizemicrospheres of gas which would be detectable by ultrasound imagingfollowing localization at the site of tissue injury.

Therefore, imaging agents based on MMP inhibitors would be extremelyuseful in the detection, staging and monitoring of cardiovasculardiseases such as atherosclerosis (especially unstable arterial plaque)and various cardiomyopathies including congestive heart failure.Compounds of the present invention, which localize in areas of MMPactivity in the heart, will allow detection and localization of thesecardiac diseases which are associated with altered MMP levels relativeto normal myocardial tissue.

These imaging agents, whether for gamma scintigraphy, positron emissiontomography, MRI, ultrasound or x-ray image enhancement, have utility todetect and monitor changes in cardiovascular diseases over time. Sincethe degree of overexpression of MMPs is related to the degradation ofcardiac or vascular tissue (Rohde L E, Aikawa M, Cheng G C, et al., JACC1999; 33: 835-842) it is possible to assess the severity and currentactivity of cardiovascular disease lesions (i.e. plaques) byquantitating the degree of localization of these imaging agents at thediseased sites of interest. Moreover, with these imaging agents it ispossible to monitor changes in MMP activity associated with theinstitution of pharmaceutical therapies which slow the progression orcause a reversal of atheroschlerotic changes in the vascular system or areversal of myocardial degradation associated with congestive heartfailure.

Therefore, it can be appreciated that the imaging of MMPs in the heartwould be generally useful for the detection, localization and monitoringthe progression/regression of a variety of cardiac diseases which areassociated with alterations in the MMP content of cardiac tissues.

SUMMARY OF THE INVENTION

It is one object of the present invention to provide imaging agents forcardiovascular pathologies associated with extracellular matrixdegradation, such as atherosclerosis, heart failure, and restenosis,comprised of matrix metalloproteinase inhibiting compounds conjugated toan imageable moiety, such as a gamma ray or positron emittingradioisotope, a magnetic resonance imaging contrast agent, an X-raycontrast agent, or an ultrasound contrast agent.

Another aspect of the present invention are diagnostic kits for thepreparation of radiopharmaceuticals useful as imaging agents forcardiovascular pathologies associated with extracellular matrixdegradation, such as atherosclerosis, heart failure, and restenosis.Diagnostic kits of the present invention comprise one or more vialscontaining the sterile, non-pyrogenic, formulation comprised of apredetermined amount of a reagent of the present invention, andoptionally other components such as one or two ancillary ligands,reducing agents, transfer ligands, buffers, lyophilization aids,stabilization aids, solubilization aids and bacteriostats. The inclusionof one or more optional components in the formulation will frequentlyimprove the ease of synthesis of the radiopharmaceutical by thepracticing end user, the ease of manufacturing the kit, the shelf-lifeof the kit, or the stability and shelf-life of the radiopharmaceutical.The inclusion of one or two ancillary ligands is required for diagnostickits comprising reagent comprising a hydrazine or hydrazone bondingmoiety. The one or more vials that contain all or part of theformulation can independently be in the form of a sterile solution or alyophilized solid.

Another aspect of the present invention contemplates a method of imagingcardiovascular pathologies associated with extracellular matrixdegradation, such as atherosclerosis, heart failure, and restenosis in apatient involving: (1) synthesizing a diagnostic radiopharmaceutical ofthe present invention, using a reagent of the present invention, capableof localizing at the loci of the cardiovascular pathology; (2)administering said radiopharmaceutical to a patient by injection orinfusion; (3) imaging the patient using planar or SPECT gammascintigraphy, or positron emission tomography.

Another aspect of the present invention contemplates a method of imagingcardiovascular pathologies associated with extracellular matrixdegradation, such as atherosclerosis, heart failure, and restenosis in apatient involving: (1) administering a paramagneticmetallopharmaceutical of the present invention capable of localizing theloci of the cardiovascular pathology to a patient by injection orinfusion; and (2) imaging the patient using magnetic resonance imaging.

Another aspect of the present invention contemplates a method of imagingcardiovascular pathologies associated with extracellular matrixdegradation, such as atherosclerosis, heart failure, and restenosis in apatient involving: (1) administering a X-ray contrast agent of thepresent invention capable of localizing in tumors to a patient byinjection or infusion; and (2) imaging the patient using X-ray computedtomography.

Another aspect of the present invention contemplates a method of imagingcardiovascular pathologies associated with extracellular matrixdegradation, such as atherosclerosis, heart failure, and restenosis in apatient involving: (1) administering a ultrasound contrast agent of thepresent invention capable of localizing in tumors to a patient byinjection or infusion; and (2) imaging the patient using sonography.

DETAILED DESCRIPTION

Thus the present invention includes the following embodiments:

(1) A diagnostic agent comprising a diagnostic metal and a compound,wherein the compound comprises:

-   i) 1-10 targeting moieties;-   ii) a chelator; and-   iii) 0-1 linking groups between the targeting moiety and chelator;-   wherein the targeting moiety is a matrix metalloproteinase    inhibitor; and-   wherein the chelator is capable of conjugating to the diagnostic    metal.

(2) A diagnostic agent according to embodiment 1, wherein the targetingmoiety is a matrix metalloproteinase inhibitor having an inhibitoryconstant K_(i) of <1000 nM.

(3) A diagnostic agent according to any of embodiments 1-2, wherein thetargeting moiety is a matrix metalloproteinase inhibitor having aninhibitory constant K_(i) of <100 nM.

(4) A diagnostic agent according to any of embodiments 1-3, comprising1-5 targeting moieties.

(5). A diagnostic agent according to any of embodiments 1-4, comprisingone targeting moiety.

(6) A diagnostic agent any of embodiments 1-5, wherein the targetingmoiety is an inhibitor of one or more matrix metalloproteinases selectedfrom the group consisting of MMP-1, MMP-2, MMP-3, MMP-9, and MMP-14.

(7) A diagnostic agent of any one of embodiments 1-6, wherein thetargeting moiety is an inhibitor of one or more matrixmetalloproteinases selected from the group consisting of MMP-2, MMP-9,and MMP-14.

(8) A diagnostic agent according to any one of embodiments 1-7 whereinthe targeting moiety is a matrix metalloproteinase inhibitor of theformulae (Ia) or (Ib):

-   R¹ is independently OH or —CH₂SH;-   R¹ is independently selected at each occurrence from the group: H,    OH, C₁₋₃ alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl, and heterocycle-S—CH₂—;-   R² is independently C₁₋₂₀ alkyl;-   X is independently C═O or SO₂, provided when X is C═O, R³ is    and when X is SO₂, R³ is independently selected from the group: aryl    substituted with 0-2 R⁶, and heterocycle substituted with 0-2 R⁶;-   R⁴ is independently selected at each occurrence from the group: C₁₋₆    alkyl, phenyl, and benzyl;-   R⁵ is independently at each occurrence from the group: NH(C₁₋₆    alkyl), NH-phenyl, and NH-heterocycle; wherein said alkyl, phenyl    and heterocycle groups are optionally substituted with a bond to the    linking group or a bond to the chelator;-   R⁶ is independently aryloxy substituted with 0-3 R⁷;-   R⁷ is independently halogen or methoxy;-   or alternatively,-   R¹ and R⁴ may be taken together to form a bridging group of the    formula —(CH₂)₃—O-phenyl-CH₂—, optionally substituted with a bond to    the linking group or a bond to the chelator;-   or alternatively,-   R¹ and R² may be taken together to form a bridging group of the    formula —(CH₂)₃—NH—, optionally substituted with a bond to the    linking group or a bond to the chelator; or-   R¹ and R² taken together with the nitrogen and carbon atom through    which they are attached form a C₅₋₇ atom saturated ring system    substituted with one or more substituents selected from the group    consisting of: a bond to Ln, a bond to Ch, and —C(═O)—NR²⁹R³⁰;-   R⁸ is independently at each occurrence OH or phenyl, optionally    substituted with a bond to the linking group or a bond to the    chelator, provided that when R⁸ is phenyl, R¹⁰ is    —C(═O)—CR¹²—NH—CH(CH₃)—COOH;-   R⁹ and R⁹′ are independently H, C₁₋₆ alkyl optionally substituted    with a bond to the linking group or a bond to the chelator, or are    taken together with the carbon atom to which R⁹ and R⁹′ are attached    to form a 5-7 atom saturated, partially unsaturated or aromatic ring    system containing 0-3 heteroatoms selected from O, N, SO₂ and S,    said ring system substituted with R⁶ and optionally substituted with    a bond to the linking group or a bond to the chelator;-   R¹⁰ and R¹¹ are independently H, or C₁₋₆ alkyl optionally    substituted with a bond to the linking group or a bond to the    chelator, or are taken together with the nitrogen atom to which they    are attached to form a 5-7 atom saturated, partially unsaturated or    aromatic ring system containing 0-3 heteroatoms selected from O, N,    SO₂ and S, said ring system optionally substituted with 0-3 R²⁷, a    bond to the linking group or a bond to the chelator;-   or alternatively,-   R⁹ and R¹⁰ are taken together with the carbon atom to which they are    attached to form a 5-7 atom saturated, partially unsaturated or    aromatic ring system containing 0-3 heteroatoms selected from O, N,    SO₂ and S, said ring system optionally substituted with a bond to    the linking group or a bond to the chelator; and-   R¹² is independently C₁₋₂₀ alkyl;-   R²⁷ is ═O, C1-4 alkyl, or phenyl substituted with R²⁸;-   R²⁸ is a phenoxy group substituted with 0-2 OCH₃ groups;-   R²⁹ and R³⁰ taken together with the nitrogen atom through which they    are attached form a C5-7 atom saturated ring system substituted with    R³¹; and-   R³¹ is a benzyloxy group substituted with C1-4 alkyl.

(9). A diagnostic agent according to any one of embodiments 1-8 whereinthe targeting moiety is a matrix metalloproteinase inhibitor of theformulae (Ia) or (Ib):

-   R is OH;-   R¹ is independently selected at each occurrence from the group: H,    OH, C₁₋₃ alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl, and heterocycle-S—CH₂—;-   R² is independently C₁₋₆ alkyl;-   X is C═O;-   R⁴ is independently selected at each occurrence from the group: C₁₋₆    alkyl, phenyl, and benzyl;-   R⁵ is independently at each occurrence from the group: NH(C₁₋₆    alkyl), NH-phenyl, and NH-heterocycle; wherein said alkyl, phenyl    and heterocycle groups are optionally substituted with a bond to the    linking group or a bond to the chelator;-   R⁶ is independently aryloxy substituted with 0-3 R⁷;-   R⁷ is independently halogen or methoxy;-   or alternatively,-   R¹ and R⁴ may be taken together to form a bridging group of the    formula —(CH₂)₃—O-phenyl-CH₂—, optionally substituted with a bond to    the linking group or a bond to the chelator;-   or alternatively,-   R¹ and R² may be taken together to form a bridging group of the    formula —(CH₂)₃—NH—, optionally substituted with a bond to the    linking group or a bond to the chelator; or-   R¹ and R² taken together with the nitrogen and carbon atom through    which they are attached form a C₅₋₇ atom saturated ring system    substituted with one or more substituents selected from the group    consisting of: a bond to Ln, a bond to Ch, and —C(═O)—NR²⁹R³⁰;-   R⁸ is OH;-   R⁹ and R⁹′ are independently H, C₁₋₆ alkyl optionally substituted    with a bond to the linking group or a bond to the chelator, or are    taken together with the carbon atom to which R⁹ and R⁹′ are attached    to form a 5-7 atom saturated, partially unsaturated or aromatic ring    system containing 0-1 heteroatoms selected from O, N, said ring    system optionally substituted with a bond to the linking group or a    bond to the chelator;-   R¹⁰ and R¹¹ are independently H, or C₁₋₆ alkyl optionally    substituted with a bond to the linking group or a bond to the    chelator, or are taken together with the nitrogen atom to which they    are attached to form a 5-7 atom saturated, partially unsaturated or    aromatic ring system containing 0-1 heteroatoms selected from O, N,    said ring system optionally substituted with 0-3 R²⁷, a bond to the    linking group or a bond to the chelator;-   or alternatively,-   R⁹ and R¹⁰ are taken together with the carbon atom to which they are    attached to form a 5-7 atom saturated, partially unsaturated or    aromatic ring system containing 0-1 heteroatoms selected from O, N,    said ring system optionally substituted with a bond to the linking    group or a bond to the chelator; and-   R¹² is independently C₁₋₆ alkyl;-   R²⁷ is ═O, C1-4 alkyl, or phenyl substituted with R²⁸;-   R²⁸ is a phenoxy group substituted with 0-2 OCH₃ groups;-   R²⁹ and R³⁰ taken together with the nitrogen atom through which they    are attached form a C₅₋₇ atom saturated ring system substituted with    R³¹; and-   R³¹ is a benzyloxy group substituted with C1-4 alkyl.

(10). A diagnostic agent according to any one of embodiments 1-9 whereinthe targeting moiety is a matrix metalloproteinase inhibitor of theformulae (Ia) or (Ib):

wherein:

-   R is —OH;-   R² is C₁₋₆ alkyl;-   X is C═O;-   R³ is-   R¹ and R⁴ are taken together to form a bridging group of formula    —(CH₂)₃—O-phenyl-CH₂—;-   R⁵ is NH(C1-6alkyl), substituted with a bond to the linking group or    a bond to the chelator.

(11) A diagnostic agent according to any one of embodiments 1-10,wherein:

-   R is —OH;-   R⁹ is C₁ alkyl substituted with a bond to Ln;-   R¹⁰ and R¹¹ taken together with the nitrogen atom to which they are    attached form a 5 atom saturated ring system, said right system is    substituted with 0-3 R²⁷;-   R²⁷ is ═O, C1-4 alkyl, or phenyl substituted with R²⁸; and-   R²⁸ is a phenoxy group substituted with 0-2 OCH₃ groups.

(12) A diagnostic agent according to any one of embodiments 1-11 whereinthe

-   R is —OH;-   R¹ and R² taken together with the nitrogen and carbon atom through    which they are attached form a C₅₋₇ atom saturated ring system    substituted with one or more substituents selected from the group    consisting of: a bond to Ln, a bond to Ch, and —C(═O)—NR²⁹R³⁰;-   R²⁹ and R³⁰ taken together with the nitrogen atom through which they    are attached form a C₅₋₇ atom saturated ring system substituted with    R³¹; and-   R³¹ is a benzyloxy group substituted with C1-4 alkyl.

(13) A diagnostic agent according to any one of embodiments 1-12 whereinthe linking group is of the formula:((W¹)_(h)—(CR¹³R¹⁴)_(g))_(x)-(Z)_(k)-((CR^(13a)R^(14a))_(g′)—(W²)_(h′))_(x′);

-   W¹ and W² are independently selected at each occurrence from the    group: O, S, NH, NHC(═O), C(═O)NH, NR¹⁵C(═O), C(═O)NR¹⁵, C(═O),    C(═O)O, OC(═O), NHC(═S)NH, NHC(═O)NH, SO₂, SO₂NH, —(OCH₂CH₂)₇₆₋₈₄,    (OCH₂CH₂)_(s), (CH₂CH₂O)_(s′), (OCH₂CH₂CH₂)_(s″), (CH₂CH₂CH₂O)_(t),    and (aa)_(t′);-   aa is independently at each occurrence an amino acid;-   Z is selected from the group: aryl substituted with 0-3 R¹⁶, C₃₋₁₀    cycloalkyl substituted with 0-3 R¹⁶, and a 5-10 membered    heterocyclic ring system containing 1-4 heteroatoms independently    selected from N, S, and O and substituted with 0-3 R¹⁶;-   R¹³, R^(13a), R¹⁴, R^(14a), and R¹⁵ are independently selected at    each occurrence from the group: H, ═O, COOH, SO₃H, PO₃H, C₁-C₅ alkyl    substituted with 0-3 R¹⁶, aryl substituted with 0-3 R¹⁶, benzyl    substituted with 0-3 R¹⁶, and C₁-C₅ alkoxy substituted with 0-3 R¹⁶,    NHC(═O)R¹⁷, C(═O)NHR¹⁷, NHC(═O)NHR¹⁷, NHR¹⁷, R¹⁷, and a bond to the    chelator;-   R¹⁶ is independently selected at each occurrence from the group: a    bond to the chelator, COOR¹⁷, C(═O)NHR¹⁷, NHC(═O)R¹⁷, OH, NHR¹⁷,    SO₃H, PO₃H, —OPO₃H₂, —OSO₃H, aryl substituted with 0-3 R¹⁷, C₁₋₅    alkyl substituted with 0-1 R¹⁸, C₁₋₅ alkoxy substituted with 0-1    R¹⁸, and a 5-10 membered heterocyclic ring system containing 1-4    heteroatoms independently selected from N, S, and O and substituted    with 0-3 R¹⁷;-   R¹⁷ is independently selected at each occurrence from the group: H,    alkyl substituted with 0-1 R¹⁸, aryl substituted with 0-1 R¹⁸, a    5-10 membered heterocyclic ring system containing 1-4 heteroatoms    independently selected from N, S, and O and substituted with 0-1    R¹⁸, C₃₋₁₀ cycloalkyl substituted with 0-1 R¹⁸, polyalkylene glycol    substituted with 0-1 R¹⁸, carbohydrate substituted with 0-1 R¹⁸,    cyclodextrin substituted with 0-1 R¹⁸, amino acid substituted with    0-1 R¹⁸, polycarboxyalkyl substituted with 0-1 R¹⁸, polyazaalkyl    substituted with 0-1 R¹⁸, peptide substituted with 0-1 R¹⁸, wherein    the peptide is comprised of 2-10 amino acids,    3,6-O-disulfo-B-D-galactopyranosyl, bis(phosphonomethyl)glycine, and    a bond to the chelator;-   R¹⁸ is a bond to the chelator;-   k is selected from 0, 1, and 2;-   h is selected from 0, 1, and 2;-   h′ is selected from 0, 1, and 2;-   g is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   g′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   s is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   s′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   s″ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   t is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   t′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   x is selected from 0, 1, 2, 3, 4, and 5; and-   x′ is selected from 0, 1, 2, 3, 4, and 5.

(14) A diagnostic agent according to any one of embodiments 1-13 wherein

-   W¹ and W² are independently selected at each occurrence from the    group: O, NH, NHC(═O), C(═O)NH, NR¹⁵C(═O), C(═O)NR¹⁵. C(═O), C(═O)O,    OC(═O), NHC(═S)NH, NHC(═O)NH, SO₂, —(CH₂CH₂O)₇₆₋₈₄—, (OCH₂CH₂)_(s),    (CH₂CH₂O)_(s′), (OCH₂CH₂CH₂)_(s″), (CH₂CH₂CH₂O)_(t), and (aa)_(t′);-   aa is independently at each occurrence an amino acid;-   Z is selected from the group: aryl substituted with 0-1 R¹⁶, C₃₋₁₀    cycloalkyl substituted with 0-1 R¹⁶, and a 5-10 membered    heterocyclic ring system containing 1-4 heteroatoms independently    selected from N, S, and O and substituted with 0-1 R¹⁶;-   R¹³, R^(13a), R¹⁴, R^(14a), and R¹⁵ are independently selected at    each occurrence from the group: H, ═O, COOH, SO₃H, C₁-C₅ alkyl    substituted with 0-1 R¹⁶, aryl substituted with 0-1 R¹⁶, benzyl    substituted with 0-1 R¹⁶, and C₁-C₅ alkoxy substituted with 0-1 R¹⁶,    NHC(═O)R¹⁷, C(═O)NHR¹⁷, NHC(═O)NHR¹⁷, NHR¹⁷, R¹⁷, and a bond to the    chelator;-   k is 0 or 1;-   s is selected from 0, 1, 2, 3, 4, and 5;-   s′ is selected from 0, 1, 2, 3, 4, and 5;-   s″ is selected from 0, 1, 2, 3, 4, and 5; and-   t is selected from 0, 1, 2, 3, 4, and 5.

(15) A diagnostic agent according to embodiment 13 wherein wherein:

-   W¹ is C(═O)NR¹⁵;-   h is 1;-   g is 3;-   R¹³ and R¹⁴ are independently H;-   x is 1;-   k is 0;-   g′ is 0;-   h′ is 1;-   W² is NH; and-   x′ is 1.

(16) A diagnostic agent according to embodiment 13 wherein

-   x is 0;-   k is 1;-   Z is aryl substituted with 0-3 R¹⁶;-   g′ is 1;-   W² is NH;-   R^(13a) and R^(14a) are independently H;-   h′ is 1; and-   x′ is 1.

(17) A diagnostic agent according to embodiment 13 wherein

-   W¹ is C(═O)NR¹⁵;-   h is 1;-   g is 2;-   R¹³ and R¹⁴ are independently H;-   x is 1;-   k is 0;-   g′ is 1;-   R^(13a) and R^(14a) are independently H; or C1-5 alkyl substituted    with 0-3 R¹⁶;-   R¹⁶ is SO₃H;-   W² is NHC(═O) or NH;-   h′ is 1; and-   x′ is 2.

(18). A diagnostic agent according to embodiment 13 wherein

-   W¹ is C(═O)NH;-   h is 1;-   g is 3;-   R¹³ and R¹⁴ are independently H;-   k is 0;-   g′ is 0;-   x is 1;-   W² is —NH(C═O)— or —(OCH₂CH₂)₇₆₋₈₄—;-   h′ is 2; and-   x′ is 1.

(19) A diagnostic agent according to embodiment 13 wherein

-   x is 0;-   k is 0;-   g′ is 3;-   h′ is 1;-   W² is NH; and-   x′ is 1.

(20) A diagnostic agent according to embodiment 13 wherein

-   x is 0;-   Z is aryl substituted with 0-3 R¹⁶;-   k is 1;-   g′ is 1;-   R^(13a)R^(14a) are independently H;-   W² is NHC(═O) or —(OCH₂CH₂)₇₆₋₈₄—; and-   x′ is 1.

(21) A diagnostic agent according to embodiment 13 wherein

-   W¹ is C═O;-   g is 2;-   R¹³ and R¹⁴ are independently H;-   k is 0;-   g′ is 0;-   h′ is 1;-   W² is NH; and-   x′ is 1.

(22) A compound according to embodiment 1 wherein the linking group isabsent.

(23) A diagnostic agent according to any one of embodiments 1-22 whereinthe chelator is a metal bonding unit having a formula selected from thegroup:

-   A¹, A², A³, A⁴, A⁵, A⁶, A⁷, and A⁸ are independently selected at    each occurrence from the group: N, NR²⁶, NR¹⁹, NR¹⁹R²⁰, S, SH,    —S(Pg), 0, OH, PR¹⁹, PR¹⁹R²⁰, —O—P(O)(R²¹)—O—, P(O)R²¹R²², a bond to    the targeting moiety and a bond to the linking group;-   Pg is a thiol protecting group;-   E¹, E², E³, E⁴, E⁵, E⁶, E⁷, and E⁸ are independently a bond, CH, or    a spacer group independently selected at each occurrence from the    group: C₁-C₁₆ alkyl substituted with 0-3 R²³, aryl substituted with    0-3 R²³, C₃₋₁₀ cycloalkyl substituted with 0-3 R²³,    heterocyclo-C₁₋₁₀ alkyl substituted with 0-3 R²³, wherein the    heterocyclo group is a 5-10 membered heterocyclic ring system    containing 1-4 heteroatoms independently selected from N, S, and O,    C₆₋₁₀ aryl-C₁₋₁₀ alkyl substituted with 0-3 R²³, C₁₋₁₀ alkyl-C₆₋₁₀    aryl-substituted with 0-3 R²³, and a 5-10 membered heterocyclic ring    system containing 1-4 heteroatoms independently selected from N, S,    and O and substituted with 0-3 R²³;-   R¹⁹ and R²⁰ are each independently selected from the group: a bond    to the linking group, a bond to the targeting moiety, hydrogen,    C₁-C₁₀ alkyl substituted with 0-3 R²³, aryl substituted with 0-3    R²³, C₁₋₁₀ cycloalkyl substituted with 0-3 R²³, heterocyclo-C₁₋₁₀    alkyl substituted with 0-3 R²³, wherein the heterocyclo group is a    5-10 membered heterocyclic ring system containing 1-4 heteroatoms    independently selected from N, S, and O, C₆₋₁₀ aryl-C₁₋₁₀ alkyl    substituted with 0-3 R²³, C₁₋₁₀ alkyl-C₆₋₁₀ aryl-substituted with    0-3 R²³, a 5-10 membered heterocyclic ring system containing 1-4    heteroatoms independently selected from N, S, and O and substituted    with 0-3 R²³, and an electron, provided that when one of R¹⁹ or R²⁰    is an electron, then the other is also an electron;-   R²¹ and R²² are each independently selected from the group: a bond    to the linking group, a bond to the targeting moiety, —OH, C₁-C₁₀    alkyl substituted with 0-3 R²³, C₁-C₁₀ alkyl substituted with 0-3    R²³, aryl substituted with 0-3 R²³, C₃₋₁₀ cycloalkyl substituted    with 0-3 R²³, heterocyclo-C₁₋₁₀ alkyl substituted with 0-3 R²³,    wherein the heterocyclo group is a 5-10 membered heterocyclic ring    system containing 1-4 heteroatoms independently selected from N, S,    and O, C₆₋₁₀ aryl-C₁₋₁₀ alkyl substituted with 0-3 R²³, C₁₋₁₀    alkyl-C₆₋₁₀ aryl-substituted with 0-3 R²³, and a 5-10 membered    heterocyclic ring system containing 1-4 heteroatoms independently    selected from N, S, and O and substituted with 0-3 R²³;-   R²³ is independently selected at each occurrence from the group: a    bond to the linking group, a bond to the targeting moiety, ═O, F,    Cl, Br, I, —CF₃, —CN, —CO₂R²⁴, —C(═O)R²⁴, —C(═O)N(R²⁴)₂, —CHO,    —CH₂OR²⁴, —OC(═O)R²⁴, —OC(═O)OR^(24a), —OR²⁴, —OC(═O)N(R²⁴)₂,    —NR²⁵C(═O)R²⁴, —NR²⁵C(═O)OR^(24a), —NR²⁵C(═O)N(R²⁴)₂,    —NR²⁵SO₂N(R²⁴)₂, —NR²⁵SO₂R^(24a), —SO₃H, SO₂R^(24a), —SR²⁴,    —S(═O)R^(24a), —SO₂N(R²⁴)₂, —N(R²⁴)₂, —NHC(═S)NHR²⁴, ═NOR²⁴, NO₂,    —C(═O)NHOR²⁴, —C(═O)NHNR²⁴R^(24a), —OCH₂CO₂H,    2-(1-morpholino)ethoxy, C₁-C₅ alkyl, C₂-C₄ alkenyl, C₃-C₆    cycloalkyl, C₃-C₆ cycloalkylmethyl, C₂-C₆ alkoxyalkyl, aryl    substituted with 0-2 R²⁴, and a 5-10 membered heterocyclic ring    system containing 1-4 heteroatoms independently selected from N, S,    and O; and-   wherein at least one of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸ or R²³ is a    bond to the linking group or targeting moiety;-   R²⁴, R^(24a), and R²⁵ are independently selected at each occurrence    from the group: a bond to the linking group, a bond to the targeting    moiety, H, C₁-C₆ alkyl, phenyl, benzyl, C₁-C₆ alkoxy, halide, nitro,    cyano, and trifluoromethyl; and-   R²⁶ is a co-ordinate bond to a metal or a hydrazine protecting    group; or a pharmaceutically acceptable salt thereof.

(24) A diagnostic agent according to any one of embodiments 1-23wherein:

-   A¹, A², A³, A⁴, A⁵, A⁶, A⁷, and A⁸ are independently selected at    each occurrence from the group: NR¹⁹, NR¹⁹R²⁰, S, SH, OH, a bond to    the targeting moiety and a bond to the linking group;-   E¹, E², E³, E⁴, E⁵, E⁶, E⁷, and E⁸ are independently a bond, CH, or    a spacer group independently selected at each occurrence from the    group: C₁-C₁₀ alkyl substituted with 0-3 R²³, aryl substituted with    0-3 R²³, C₃₋₁₀ cycloalkyl substituted with 0-3 R²³, and a 5-10    membered heterocyclic ring system containing 1-4 heteroatoms    independently selected from N, S, and O and substituted with 0-3    R²³;-   wherein at least one of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸ and R²³ is a    bond to the linking group or the targeting moiety;-   R¹⁹, and R²⁰ are each independently selected from the group: a bond    to the targeting moiety, a bond to the linking group, hydrogen,    C₁-C₁₀ alkyl substituted with 0-3 R²³, aryl substituted with 0-3    R²³, a 5-10 membered heterocyclic ring system containing 1-4    heteroatoms independently selected from N, S, and O and substituted    with 0-3 R²³, and an electron, provided that when one of R¹⁹ or R²⁰    is an electron, then the other is also an electron;-   R²³ is independently selected at each occurrence from the group: a    bond to the targeting moiety, a bond to the linking group, ═O, F,    Cl, Br, I, —CF₃, —CN, —CO₂R²⁴, —C(═O)R²⁴, —C(═O)N(R²⁴)₂, —CH₂OR²⁴,    —OC(═O)R²⁴, —OC(═O)OR^(24a), —OR²⁴, —OC(═O)N(R²⁴)₂, —NR²⁵C(═O)R²⁴,    —NR²⁵C(═O)OR^(24a), —NR²⁵C(═O)N(R²⁴)₂, —NR²⁵SO₂N(R²⁴)₂,    —NR²⁵SO₂R^(24a), —SO₃H, SO₂R^(24a), S(═O)R^(24a), —SO₂N(R²⁴)₂,    —N(R²⁴)₂, —NHC(═S)NHR²⁴, ═NOR¹⁸, —C(═O)NHNR¹⁸R^(18a), —OCH₂CO₂H, and    2-(1-morpholino)ethoxy; and-   R²⁴, R^(24a), and R²⁵ are independently selected at each occurrence    from the group: a bond to the linking group, H, and C₁-C₆ alkyl.

(25) A diagnostic agent according to any one of embodiments 1-24 whereinthe chelator is of the formula:

-   A¹ is a bond to the linking group;-   A², A⁴, and A⁶ are each N;-   A³, A⁵, A⁷ and A⁸ are each OH;-   E¹, E², and E⁴ are C₂ alkyl;-   E³, E⁵, E⁷, and E⁸ are C₂ alkyl substituted with 0-1 R²³;-   R²³ is ═O.

(26) A diagnostic agent according to any one of embodiments 1-25 whereinthe chelator is of the formula:

-   Ch is    wherein:-   A5 is a bond to Ln;-   A¹, A³, A⁷ and A⁸ are each OH;-   A², A⁴ and A⁶ are each NH;-   E¹, E³, E⁵, E⁷, and E⁸ are C₂ alkyl substituted with 0-1 R²³;-   E², and E⁴, are C₂ alkyl;-   R²³ is ═O.

(27) A diagnostic agent according to any one of embodiments 1-26 whereinthe chelator is of the formula:

-   A¹, A², A³ and A⁴ are each N;-   A⁵, A⁶ and A⁸ are each OH;-   A⁷ is a bond to Ln;-   E¹, E², E³, E⁴ are each independently C₂ alkyl; and-   E⁵, E⁶, E⁷, E⁸ are each independently C₂ alkyl substituted with 0-1    R²³;-   R²³ is ═O.

(28) A diagnostic agent according to any one of embodiments 1-27 whereinthe chelator is of the formula:

-   A¹ is NR²⁶;-   R²⁶ is a co-ordinate bond to a metal or a hydrazine protecting    group;-   E¹ is a bond;-   A² is NHR¹⁹;-   R¹⁹ is a heterocycle substituted with R²³, the heterocycle being    selected from pyridine and pyrimidine;-   R²³ is selected from a bond to the linking group, C(═O)NHR²⁴ and    C(═O)R²⁴; and-   R²⁴ is a bond to the linking group.

(29) A diagnostic agent according to any one of embodiments 1-28 whereinthe chelator is of the formula:

wherein:

-   A¹ and A⁵ are each —S(Pg);-   Pg is a thiol protecting group;-   E¹ and E⁴ are C₂ alkyl substituted with 0-1 R²³;-   R²³ is ═O;-   A² and A⁴ are each —NH;-   E² is CH₂;-   E³ is C₁₋₃ alkyl substituted with 0-1 R²³;-   A³ is a bond to Ln.

(30) A diagnostic agent according to any one of embodiments 1-29 whereinthe chelator is of the formula:

wherein:

-   A¹ is a bond to Ln;-   E¹ is C₁, alkyl substituted by R²³;-   A² is NH;-   E² is C₂ alkyl substituted with 0-1R²³;-   A³ is —O—P(O)(R²¹)—O;-   E³ is C₁, alkyl;-   A⁴ and A⁵ are each —O—;-   E⁴ and E⁶ are each independently C₁₋₁₆ alkyl substituted with    0-1R²³;-   E⁵ is C₁, alkyl;-   R²¹ is —OH; and-   R²³ is ═O.

(31) A diagnostic agent according to embodiment 1 having the formula:(Q)_(d)-L_(n)-C_(h)wherein, Q is a compound of Formulae (Ia) or (Ib):

-   R is independently OH or —CH₂SH;-   R¹ is independently selected at each occurrence from the group: H,    OH, C₁₋₃ alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl, and heterocycle-S—CH₂—;-   R² is independently C₁₋₂₀ alkyl;-   X is independently C═O or SO₂, provided when X is C═O, R³ is    and when X is SO₂, R³ is independently selected from the group: aryl    substituted with 0-2 R⁶, and heterocycle substituted with 0-2 R⁶;-   R⁴ is independently selected at each occurrence from the group: C₁₋₆    alkyl, phenyl, and benzyl;-   R⁵ is independently at each occurrence from the group: NH(C₁₋₆    alkyl), NH-phenyl, and NH-heterocycle; wherein said alkyl, phenyl    and heterocycle groups are optionally substituted with a bond to    L_(n);-   R⁶ is independently aryloxy substituted with 0-3 R⁷;-   R⁷ is independently halogen or methoxy;-   or alternatively,-   R¹ and R⁴ may be taken together to form a bridging group of the    formula —(CH₂)₃—O-phenyl-CH₂—, optionally substituted with a bond to    L_(n);-   or alternatively,-   R¹ and R² may be taken together to form a bridging group of the    formula —(CH₂)₃—NH—, optionally substituted with a bond to L_(n); or-   R¹ and R² taken together with the nitrogen and carbon atom through    which they are attached form a C₅₋₇ atom saturated ring system    substituted with one or more substituents selected from the group    consisting of: a bond to Ln, a bond to Ch, and —C(═O)—NR²⁹R³⁰;-   R⁸ is independently at each occurrence OH or phenyl, optionally    substituted with a bond to L_(n), provided that when R⁸ is phenyl,    R¹⁰ is —C(═O)—CR¹²—NH—CH(CH₃)—COOH;-   R⁹ and R⁹′ are independently H, C₁₋₆ alkyl optionally substituted    with a bond to L_(n), or are taken together with the carbon atom to    which they are attached to form a 5-7 atom saturated, partially    unsaturated or aromatic ring system containing 0-3 heteroatoms    selected from O, N, SO₂ and S, said ring system substituted with R⁶    and optionally substituted with a bond to L_(n);-   R¹⁰ and R¹¹ are independently H, or C₁₋₆ alkyl optionally    substituted with a bond to L_(n), or are taken together with the    nitrogen atom to which they are attached to form a 5-7 atom    saturated, partially unsaturated or aromatic ring system containing    0-3 heteroatoms selected from O, N, SO₂ and S, said ring system    optionally substituted with 0-3 R²⁷ or a bond to L_(n);-   or alternatively,-   R⁹ and R¹⁰ are taken together with the carbon atom to which they are    attached to form a 5-7 atom saturated, partially unsaturated or    aromatic ring system containing 0-3 heteroatoms selected from O, N,    SO₂ and S, said ring system optionally substituted with a bond to    L_(n);-   R¹² is independently C₁₋₂₀ alkyl;-   d is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   L_(n) is a linking group having the formula:    ((W¹)_(h)—(CR¹³R¹⁴)_(g))_(x)-(Z)_(k)-((CR^(13a)R^(14a))_(g′)—(W²)_(h′))_(x′);-   W¹ and W² are independently selected at each occurrence from the    group: O, S, NH, NHC(═O), C(═O)NH, NR¹⁵C(═O), C(═O)NR¹⁵, C(═O),    C(═O)O, OC(═O), NHC(═S)NH, NHC(═O)NH, SO₂, SO₂NH, —(OCH₂CH₂)₇₆₋₈₄,    (OCH₂CH₂)_(s), (CH₂CH₂O)_(s′), (OCH₂CH₂CH₂)_(s″), (CH₂CH₂CH₂O)_(t),    and (aa)_(t′);-   aa is independently at each occurrence an amino acid;-   Z is selected from the group: aryl substituted with 0-3 R¹⁶, C₃₋₁₀    cycloalkyl substituted with 0-3 R¹⁶, and a 5-10 membered    heterocyclic ring system containing 1-4 heteroatoms independently    selected from N, S, and O and substituted with 0-3 R¹⁶;-   R¹³, R^(13a), R¹⁴, R^(14a), and R¹⁵ are independently selected at    each occurrence from the group: H, ═O, COOH, SO₃H, PO₃H, C₁-C₅ alkyl    substituted with 0-3 R¹⁶, aryl substituted with 0-3 R¹⁶, benzyl    substituted with 0-3 R¹⁶, and C₁-C₅ alkoxy substituted with 0-3 R¹⁶,    NHC(═O)R¹⁷, C(═O)NHR¹⁷, NHC(═O)NHR¹⁷, NHR¹⁷, R¹⁷, and a bond to    C_(h);-   R¹⁶ is independently selected at each occurrence from the group: a    bond to C_(h), COOR¹⁷, C(═O)NHR¹⁷, NHC(═O)R¹⁷, OH, NHR¹⁷, SO₃H,    PO₃H, —OPO₃H₂, —OSO₃H, aryl substituted with 0-3 R¹⁷, C₁₋₅ alkyl    substituted with 0-1 R¹⁸, C₁₋₅ alkoxy substituted with 0-1 R¹⁸, and    a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms    independently selected from N, S, and O and substituted with 0-3    R¹⁷;-   R¹⁷ is independently selected at each occurrence from the group: H,    alkyl substituted with 0-1 R¹⁸, aryl substituted with 0-1 R¹⁸, a    5-10 membered heterocyclic ring system containing 1-4 heteroatoms    independently selected from N, S, and O and substituted with 0-1    R¹⁸, C₃₋₁₀ cycloalkyl substituted with 0-1 R¹⁸, polyalkylene glycol    substituted with 0-1 R¹⁸, carbohydrate substituted with 0-1 R¹⁸,    cyclodextrin substituted with 0-1 R¹⁸, amino acid substituted with    0-1 R¹⁸, polycarboxyalkyl substituted with 0-1 R¹⁸, polyazaalkyl    substituted with 0-1 R¹⁸, peptide substituted with 0-1 R¹⁸, wherein    the peptide is comprised of 2-10 amino acids,    3,6-O-disulfo-B-D-galactopyranosyl, bis(phosphonomethyl)glycine, and    a bond to C_(h);-   R¹⁸ is a bond to C_(h);-   k is selected from 0, 1, and 2;-   h is selected from 0, 1, and 2;-   h′ is selected from 0, 1, and 2;-   g is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   g′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   s is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   s′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   s″ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   t is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   t′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   x is selected from 0, 1, 2, 3, 4, and 5;-   x′ is selected from 0, 1, 2, 3, 4, and 5;-   C_(h) is a metal bonding unit having a formula selected from the    group:-   A¹, A², A³, A⁴, A⁵, A⁶, A⁷, and A⁸ are independently selected at    each occurrence from the group: N, NR²⁶, NR¹⁹, NR¹⁹R²⁰, S, SH,    —S(Pg), 0, OH, PR¹⁹, PR¹⁹R²⁰, —O—P(O)(R²¹)—O—, P(O)R²¹R²², a bond to    the targeting moiety and a bond to the linking group;-   Pg is a thiol protecting group;-   E¹, E², E³, E⁴, E⁵, E⁶, E⁷, and E⁸ are independently a bond, CH, or    a spacer group independently selected at each occurrence from the    group: C₁-C₁₆ alkyl substituted with 0-3 R²³, aryl substituted with    0-3 R²³, C₃₋₁₀ cycloalkyl substituted with 0-3 R²³,    heterocyclo-C₁₋₁₀ alkyl substituted with 0-3 R²³, wherein the    heterocyclo group is a 5-10 membered heterocyclic ring system    containing 1-4 heteroatoms independently selected from N, S, and O,    C₆₋₁₀ aryl-C₁₋₁₀ alkyl substituted with 0-3 R²³, C₁₋₁₀ alkyl-C₆₋₁₀    aryl-substituted with 0-3 R²³, and a 5-10 membered heterocyclic ring    system containing 1-4 heteroatoms independently selected from N, S,    and O and substituted with 0-3 R²³;-   R¹⁹ and R²⁰ are each independently selected from the group: a bond    to the linking group, a bond to the targeting moiety, hydrogen,    C₁-C₁₀ alkyl substituted with 0-3 R²³, aryl substituted with 0-3    R²³, C₁₋₁₀ cycloalkyl substituted with 0-3 R²³, heterocyclo-C₁₋₁₀    alkyl substituted with 0-3 R²³, wherein the heterocyclo group is a    5-10 membered heterocyclic ring system containing 1-4 heteroatoms    independently selected from N, S, and O, C₆₋₁₀ aryl-C₁₋₁₀ alkyl    substituted with 0-3 R²³, C₁₋₁₀ alkyl-C₆₋₁₀ aryl-substituted with    0-3 R²³, a 5-10 membered heterocyclic ring system containing 1-4    heteroatoms independently selected from N, S, and O and substituted    with 0-3 R²³, and an electron, provided that when one of R¹⁹ or R²⁰    is an electron, then the other is also an electron;-   R²¹ and R²² are each independently selected from the group: a bond    to the linking group, a bond to the targeting moiety, —OH, C₁-C₁₀    alkyl substituted with 0-3 R²³, C₁-C₁₀ alkyl substituted with 0-3    R²³, aryl substituted with 0-3 R²³, C₃₋₁₀ cycloalkyl substituted    with 0-3 R²³, heterocyclo-C₁₋₁₀ alkyl substituted with 0-3 R²³,    wherein the heterocyclo group is a 5-10 membered heterocyclic ring    system containing 1-4 heteroatoms independently selected from N, S,    and O, C₆₋₁₀ aryl-C₁₋₁₀ alkyl substituted with 0-3 R²³, C₁₋₁₀    alkyl-C₆₋₁₀ aryl-substituted with 0-3 R²³, and a 5-10 membered    heterocyclic ring system containing 1-4 heteroatoms independently    selected from N, S, and O and substituted with 0-3 R²³;-   R²³ is independently selected at each occurrence from the group: a    bond to the linking group, a bond to the targeting moiety, ═O, F,    Cl, Br, I, —CF₃, —CN, —CO₂R²⁴, —C(═O)R²⁴, —C(═O)N(R²⁴)₂, —CHO,    —CH₂OR²⁴, —OC(═O)R²⁴, —OC(═O)OR^(24a), —OR²⁴, —OC(═O)N(R²⁴)₂,    —NR²⁵C(═O)R²⁴, —NR²⁵C(═O)OR^(24a), —NR²⁵C(═O)N(R²⁴)₂,    —NR²⁵SO₂N(R²⁴)₂, —NR²⁵SO₂R^(24a), —SO₃H, SO₂R^(24a), —SR²⁴,    —S(═O)R^(24a), —SO₂N(R²⁴)₂, —N(R²⁴)₂, —NHC(═S)NHR²⁴, ═NOR²⁴, NO₂,    —C(═O)NHOR²⁴, —C(═O)NHNR²⁴R^(24a), —OCH₂CO₂H,    2-(1-morpholino)ethoxy, C₁-C₅ alkyl, C₂-C₄ alkenyl, C₃-C₆    cycloalkyl, C₃-C₆ cycloalkylmethyl, C₂-C₆ alkoxyalkyl, aryl    substituted with 0-2 R²⁴, and a 5-10 membered heterocyclic ring    system containing 1-4 heteroatoms independently selected from N, S,    and O; and-   wherein at least one of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸ or R²³ is a    bond to the linking group or targeting moiety;-   R²⁴, R^(24a), and R²⁵ are independently selected at each occurrence    from the group: a bond to the linking group, a bond to the targeting    moiety, H, C₁-C₆ alkyl, phenyl, benzyl, C₁-C₆ alkoxy, halide, nitro,    cyano, and trifluoromethyl; and-   R²⁶ is a co-ordinate bond to a metal or a hydrazine protecting    group; or-   a pharmaceutically acceptable salt thereof.

(32) A diagnostic agent according to Embodiment 31, wherein:

-   h′ is 1;-   W² is NH; and-   x′ is 1.

(33) A diagnostic agent according to any one of embodiments 1-, wherein:

-   x is 0;-   Z is aryl substituted with 0-3 R¹⁶;-   k is 1;-   g′ is 1;-   R^(13a)R^(14a) are independently H;-   W² is NHC(═O) or —(OCH₂CH₂)₇₆₋₈₄—; and-   x′ is 1.

(34) A diagnostic agent according to any one of embodiments 31-33,wherein:

-   W¹ is C═O;-   g is 2;-   R¹³ and R¹⁴ are independently H;-   k is 0;-   g′ is 0;-   h′ is 1;-   W² is NH; and-   x′ is 1.

(35) A diagnostic agent according to to any one of embodiments 31-34,wherein:

-   2-{[5-(3-{2-[(6-Hydroxycarbamoyl-7-isobutyl-8-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10-carbonyl)-amino]-acetylamino}-propylcarbamoyl)-pyridin-2-yl]-hydrazonomethyl}-benzenesulfonic    acid;-   2-{[5-(4-{[(6-Hydroxycarbamoyl-7-isobutyl-8-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10-carbonyl)-amino]-methyl}-benzylcarbamoyl)-pyridin-2-yl]-hydrazonomethyl}-benzenesulfonic    acid;-   2-[7-({N-[3-(2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}acetylamino)propyl]carbamoyl}methyl)-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl]acetic    acid;-   2-{7-[(N-{[4-({[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-carbonylamino}methyl)phenyl]methyl}carbamoyl)methyl]-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl}acetic    acid;-   2-(7-{[N-(1-{N-[3-(2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}acetylamino)propyl]carbamoyl}-2-sulfoethyl)carbamoyl]methyl}-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl)acetic    acid;-   2-[7-({N-[1-(N-{[4-({[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-carbonylamino}methyl)phenyl]methyl}carbamoyl)-2-sulfoethyl]carbamoyl}methyl)-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl]acetic    acid;-   2-({2-[({N-[3-(2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}acetylamino)propyl]carbamoyl}methyl)(carboxymethyl)amino}ethyl){2-[bis(carboxymethyl)amino]ethyl}amino]acetic    acid;-   2-[(2-{[(N-{[4-({[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-carbonylamino}methyl)phenyl]methyl}carbamoyl)methyl](carboxymethyl)amino}ethyl){2-[bis(carboxymethyl)amino]ethyl}amino]acetic    acid;-   N-[3-(2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}acetylamino)propyl]-4,5-bis[2-(ethoxyethylthio)acetylamino]pentanamide;-   N-{[4-({[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}methyl)-phenyl]methyl}-4,5-bis[2-(ethoxyethylthio)acetylamino]-pentanamide;-   1-(1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamino)-α,ω-dicarbonylPEG₃₄₀₀-2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}-N-(3-aminopropyl)acetamide;-   1-(1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamino)-α,ω-dicarbonylPEG₃₄₀₀-[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-N-{[4-(aminomethyl)phenyl]methyl}carboxamide    conjugate;-   2-[2-({5-[N-(5-(N-hydroxycarbamoyl)(5R)-5-{3-[4-(3,4-dimethoxyphenoxy)phenyl]-3-methyl-2-oxopyrrolidinyl}pentyl)carbamoyl](2-pyridyl)}amino)(1Z)-2-azavinyl]benzenesulfonic    acid;-   2-(2-{[5-(N-{3-[3-(N-hydroxycarbamoyl)(4S)-4-({4-[(4-methylphenyl)methoxy]piperidyl}carbonyl)piperidyl]-3-oxopropyl}carbamoyl)(2-pyridyl)]amino}(1Z)-2-azavinyl)benzenesulfonic    acid; and

(36) A diagnostic agent according to to any one of embodiments 31-35wherein the diagnostic metal is selected from the group consisting of: aparamagnetic metal, a ferromagnetic metal, a gamma-emittingradioisotope, or an x-ray absorber.

(37) A diagnostic agent according to to any one of embodiments 31-36wherein the diagnostic metal is radioisotope selected from the groupconsisting of ^(99m)Tc, ⁹⁵Tc, ¹¹¹In, ⁶²Cu, ⁶⁴Cu, ⁶⁷Ga, and ⁶⁸Ga.

(38). A diagnostic agent according to to any one of embodiments 31-37further comprising a first ancillary ligand and a second ancillaryligand capable of stabilizing the radioisotope.

(39) A diagnostic agent according to Embodiment 37, wherein theradioisotope is ^(99m)Tc.

(40) A diagnostic agent according to Embodiment 37, wherein theradioisotope is ¹¹¹In.

(41) A diagnostic agent according to embodiment 36 wherein theparamagnetic metal ion is selected from the group consisting of Gd(III),Dy(III), Fe(III), and Mn(II).

(42). A diagnostic agent according to embodiment 36 wherein the x-rayabsorber is a metal is selected from the group consisting of: Re, Sm,Ho, Lu, Pm, Y, Bi, Pd, Gd, La, Au, Au, Yb, Dy, Cu, Rh, Ag, and Ir.

(43) A diagnostic composition comprising a compound according to any oneof embodiments 1-42 or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier.

(44) A kit comprising a compound of to any one of embodiments 1-42, or apharmaceutically acceptable salt form thereof and a pharmaceuticallyacceptable carrier.

(45) A kit according to Embodiment 44, wherein the kit further comprisesone or more ancillary ligands and a reducing agent.

(46) A kit according to Embodiment 45, wherein the ancillary ligands aretricine and TPPTS.

(47) A kit according to Embodiment 45, wherein the reducing agent istin(II).

(48) A diagnostic agent comprising an echogenic gas and a compound,wherein the compound comprises:

-   i) 1-10 targeting moieties;-   ii) a surfactant (Sf); and-   iii) 0-1 linking groups between the targeting moiety and surfactant;-   wherein the targeting moiety is a matrix metalloproteinase    inhibitor; and-   wherein the surfactant is capable of forming an echogenic gas filled    lipid sphere or microbubble.

(49) A diagnostic agent according to embodiment 48, wherein thetargeting moiety is a matrix metalloproteinase inhibitor having aninhibitory constant K_(i) of <1000 nM.

(50) A diagnostic agent according to any one of embodiments 48-49,wherein the targeting moiety is a matrix metalloproteinase inhibitorhaving an inhibitory constant K_(i) of <100 nM.

(51) A diagnostic agent according to embodiment 48, comprising 1-5targeting moieties.

(52). A diagnostic agent according to embodiment 48, comprising onetargeting moiety.

(53) A diagnostic agent according to any one of embodiments 48-52,wherein the targeting moiety is an inhibitor of one or more matrixmetalloproteinases selected from the group consisting of MMP-1, MMP-2,MMP-3, MMP-9, and MMP-14.

(54) A diagnostic agent according to any one of embodiments 48-53,wherein the targeting moiety is an inhibitor of one or more matrixmetalloproteinases selected from the group consisting of MMP-2, MMP-9,and MMP-14.

(55) A diagnostic agent according to embodiment 48, wherein thetargeting moiety is of the formulae (Ia) or (Ib):

-   R is independently OH or —CH₂SH;-   R¹ is independently selected at each occurrence from the group: H,    OH, C₁₋₃ alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl, and heterocycle-S—CH₂—;-   R² is independently C₁₋₂₀ alkyl;-   X is independently C═O or SO₂, provided when X is C═O, R³ is    and when X is SO₂, R³ is independently selected from the group: aryl    substituted with 0-2 R⁶, and heterocycle substituted with 0-2 R⁶;-   R⁴ is independently selected at each occurrence from the group: C₁₋₆    alkyl, phenyl, and benzyl;-   R⁵ is independently at each occurrence from the group: NH(C₁₋₆    alkyl), NH-phenyl, and NH-heterocycle; wherein said alkyl, phenyl    and heterocycle groups are optionally substituted with a bond to the    linking group or a bond to the surfactant;-   R⁶ is independently aryloxy substituted with 0-3 R⁷;-   R⁷ is independently halogen or methoxy;-   or alternatively,-   R¹ and R⁴ may be taken together to form a bridging group of the    formula —(CH₂)₃—O-phenyl-CH₂—, optionally substituted with a bond to    the linking group or a bond to the surfactant;-   or alternatively,-   R¹ and R² may be taken together to form a bridging group of the    formula —(CH₂)₃—NH—, optionally substituted with a bond to the    linking group or a bond to the surfactant; or-   R¹ and R² taken together with the nitrogen and carbon atom through    which they are attached form a C₅₋₇ atom saturated ring system    substituted with one or more substituents selected from the group    consisting of: a bond to Ln, a bond to Sf, and —C(═O)—NR²⁹R³⁰;-   R⁸ is independently at each occurrence OH or phenyl, optionally    substituted with a bond to the linking group or a bond to the    surfactant, provided that when R⁸ is phenyl, R¹⁰ is    —C(═O)—CR¹²—NH—CH(CH₃)—COOH;-   R⁹ and R⁹′ are independently H, C₁₋₆ alkyl optionally substituted    with a bond to the linking group or a bond to the surfactant, or are    taken together with the carbon atom to which R⁹ and R⁹′ are attached    to form a 5-7 atom saturated, partially unsaturated or aromatic ring    system containing 0-3 heteroatoms selected from O, N, SO₂ and S,    said ring system substituted with R⁶ and optionally substituted with    a bond to the linking group or a bond to the surfactant;-   R¹⁰ and R¹¹ are independently H, or C₁₋₆ alkyl optionally    substituted with a bond to the linking group or a bond to the    surfactant, or are taken together with the nitrogen atom to which    they are attached to form a 5-7 atom saturated, partially    unsaturated or aromatic ring system containing 0-3 heteroatoms    selected from O, N, SO₂ and S, said ring system optionally    substituted with 0-3 R²⁷, a bond to the linking group or a bond to    the surfactant;-   or alternatively,-   R⁹ and R¹⁰ are taken together with the carbon atom to which they are    attached to form a 5-7 atom saturated, partially unsaturated or    aromatic ring system containing 0-3 heteroatoms selected from O, N,    SO₂ and S, said ring system optionally substituted with a bond to    the linking group or a bond to the surfactant; and-   R¹² is independently C₁₋₂₀ alkyl;-   R²⁷ is ═O, C1-4 alkyl, or phenyl substituted with R²⁸;-   R²⁸ is a phenoxy group substituted with 0-2 OCH₃ groups;-   R²⁹ and R³⁰ taken together with the nitrogen atom through which they    are attached form a C5-7 atom saturated ring system substituted with    R³¹; and-   R³¹ is a benzyloxy group substituted with C1-4 alkyl.

(56) A diagnostic agent according to embodiment 55 wherein wherein thetargeting moiety is a matrix metalloproteinase inhibitor of the formulae(Ia) or (Ib):

-   R is OH;-   R¹ is independently selected at each occurrence from the group: H,    OH, C₁₋₃ alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl, and heterocycle-S—CH₂—;-   R² is independently C₁₋₆ alkyl;-   X is C═O;-   R⁴ is independently selected at each occurrence from the group: C₁₋₆    alkyl, phenyl, and benzyl;-   R⁵ is independently at each occurrence from the group: NH(C₁₋₆    alkyl), NH-phenyl, and NH-heterocycle; wherein said alkyl, phenyl    and heterocycle groups are optionally substituted with a bond to the    linking group or a bond to the surfactant;-   R⁶ is independently aryloxy substituted with 0-3 R⁷;-   R⁷ is independently halogen or methoxy;-   or alternatively,-   R¹ and R⁴ may be taken together to form a bridging group of the    formula —(CH₂)₃—O-phenyl-CH₂—, optionally substituted with a bond to    the linking group or a bond to the surfactant;-   or alternatively,-   R¹ and R² may be taken together to form a bridging group of the    formula —(CH₂)₃—NH—, optionally substituted with a bond to the    linking group or a bond to the surfactant; or-   R¹ and R² taken together with the nitrogen and carbon atom through    which they are attached form a C₅₋₇ atom saturated ring system    substituted with one or more substituents selected from the group    consisting of: a bond to Ln, a bond to Sf, and —C(═O)—NR²⁹R³⁰;-   R⁸ is OH;-   R⁹ and R⁹′ are independently H, C₁₋₆ alkyl optionally substituted    with a bond to the linking group or a bond to the surfactant, or are    taken together with the carbon atom to which R⁹ and R⁹′ are attached    to form a 5-7 atom saturated, partially unsaturated or aromatic ring    system containing 0-1 heteroatoms selected from O, N, said ring    system optionally substituted with a bond to the linking group or a    bond to the surfactant;-   R¹⁰ and R¹¹ are independently H, or C₁₋₆ alkyl optionally    substituted with a bond to the linking group or a bond to the    surfactant, or are taken together with the nitrogen atom to which    they are attached to form a 5-7 atom saturated, partially    unsaturated or aromatic ring system containing 0-1 heteroatoms    selected from O, N, said ring system optionally substituted with 0-3    R²⁷, a bond to the linking group or a bond to the surfactant;-   or alternatively,-   R⁹ and R¹⁰ are taken together with the carbon atom to which they are    attached to form a 5-7 atom saturated, partially unsaturated or    aromatic ring system containing 0-1 heteroatoms selected from O, N,    said ring system optionally substituted with a bond to the linking    group or a bond to the surfactant; and-   R¹² is independently C₁₋₆ alkyl;-   R²⁷ is ═O, C1-4 alkyl, or phenyl substituted with R²⁸;-   R²⁸ is a phenoxy group substituted with 0-2 OCH₃ groups;-   R²⁹ and R³⁰ taken together with the nitrogen atom through which they    are attached form a C5-7 atom saturated ring system substituted with    R³¹; and-   R³¹ is a benzyloxy group substituted with C₁₋₄ alkyl.

(57) A diagnostic agent according to any one of embodiments 55-57wherein the targeting moiety is a matrix metalloproteinase inhibitor ofthe formulae (Ia) or (Ib):

wherein:

-   R is —OH;-   R² is C₁₋₆ alkyl;-   X is C═O;-   R³ is-   R¹ and R⁴ are taken together to form a bridging group of formula    —(CH₂)₃—O-phenyl-CH₂—;-   R⁵ is NH(C1-6alkyl), substituted with a bond to the linking group or    a bond to the surfactant.

(58) A diagnostic agent according to any one of embodiments 55-57wherein:

-   R is —OH;-   R⁹ is C₁ alkyl substituted with a bond to Ln;-   R¹⁰ and R¹¹ taken together with the nitrogen atom to which they are    attached form a 5 atom saturated ring system, said right system is    substituted with 0-3 R²⁷;-   R²⁷ is ═O, C1-4 alkyl, or phenyl substituted with R²⁸; and-   R²⁸ is a phenoxy group substituted with 0-2 OCH₃ groups.

(59) A diagnostic agent according to any one of embodiments 55-58wherein

-   R is —OH;-   R¹ and R² taken together with the nitrogen and carbon atom through    which they are attached form a C₅₋₇ atom saturated ring system    substituted with one or more substituents selected from the group    consisting of: a bond to Ln, a bond to Sf, and —C(═O)—NR²⁹R³⁰;-   R²⁹ and R³⁰ taken together with the nitrogen atom through which they    are attached form a C5-7 atom saturated ring system substituted with    R³¹; and-   R³¹ is a benzyloxy group substituted with C1-4 alkyl.

(60) A diagnostic agent according to any one of embodiments 48-59wherein the linking group is of the formula:((W¹)_(h)—(CR¹³R¹⁴)_(g))_(x)-(Z)_(k)-((CR^(13a)R^(14a))_(g′)—(W²)_(h′))_(x′);

-   W¹ and W² are independently selected at each occurrence from the    group: O, S, NH, NHC(═O), C(═O)NH, NR¹⁵C(═O), C(═O)NR¹⁵, C(═O),    C(═O)O, OC(═O), NHC(═S)NH, NHC(═O)NH, SO₂, SO₂NH, —(OCH₂CH₂)₇₆₋₈₄,    (OCH₂CH₂)_(s), (CH₂CH₂O)_(s′), (OCH₂CH₂CH₂)_(s″), (CH₂CH₂CH₂O)_(t),    and (aa)_(t′);-   aa is independently at each occurrence an amino acid;-   Z is selected from the group: aryl substituted with 0-3 R¹⁶, C₃₋₁₀    cycloalkyl substituted with 0-3 R¹⁶, and a 5-10 membered    heterocyclic ring system containing 1-4 heteroatoms independently    selected from N, S, and O and substituted with 0-3 R¹⁶;-   R¹³, R^(13a), R¹⁴, R^(14a), and R¹⁵ are independently selected at    each occurrence from the group: H, ═O, COOH, SO₃H, PO₃H, C₁-C₅ alkyl    substituted with 0-3 R¹⁶, aryl substituted with 0-3 R¹⁶, benzyl    substituted with 0-3 R¹⁶, and C₁-C₅ alkoxy substituted with 0-3 R¹⁶,    NHC(═O)R¹⁷, C(═O)NHR¹⁷, NHC(═O)NHR¹⁷, NHR¹⁷, R¹⁷, and a bond to the    surfactant;-   R¹⁶ is independently selected at each occurrence from the group: a    bond to the surfactant, COOR¹⁷, C(═O)NHR¹⁷, NHC(═O)R¹⁷, OH, NHR¹⁷,    SO₃H, PO₃H, —OPO₃H₂, —OSO₃H, aryl substituted with 0-3 R¹⁷, C₁₋₅    alkyl substituted with 0-1 R¹⁸, C₁₋₅ alkoxy substituted with 0-1    R¹⁸, and a 5-10 membered heterocyclic ring system containing 1-4    heteroatoms independently selected from N, S, and O and substituted    with 0-3 R¹⁷;-   R¹⁷ is independently selected at each occurrence from the group: H,    alkyl substituted with 0-1 R¹⁸, aryl substituted with 0-1 R¹⁸, a    5-10 membered heterocyclic ring system containing 1-4 heteroatoms    independently selected from N, S, and O and substituted with 0-1    R¹⁸, C₃₋₁₀ cycloalkyl substituted with 0-1 R¹⁸, polyalkylene glycol    substituted with 0-1 R¹⁸, carbohydrate substituted with 0-1 R¹⁸,    cyclodextrin substituted with 0-1 R¹⁸, amino acid substituted with    0-1 R¹⁸, polycarboxyalkyl substituted with 0-1 R¹⁸, polyazaalkyl    substituted with 0-1 R¹⁸, peptide substituted with 0-1 R¹⁸, wherein    the peptide is comprised of 2-10 amino acids,    3,6-O-disulfo-B-D-galactopyranosyl, bis(phosphonomethyl)glycine, and    a bond to the surfactant;-   R¹⁸ is a bond to the surfactant;-   k is selected from 0, 1, and 2;-   h is selected from 0, 1, and 2;-   h′ is selected from 0, 1, and 2;-   g is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   g′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   s is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   s′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   s″ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   t is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   t′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   x is selected from 0, 1, 2, 3, 4, and 5; and-   x′ is selected from 0, 1, 2, 3, 4, and 5.

(61) A diagnostic agent according to any one of embodiments 48-60wherein

-   W¹ and W² are independently selected at each occurrence from the    group: O, NH, NHC(═O), C(═O)NH, NR¹⁵C(═O), C(═O)NR¹⁵, C(═O), C(═O)O,    OC(═O), NHC(═S)NH, NHC(═O)NH, SO₂, —(CH₂CH₂O)₇₆₋₈₄—, (OCH₂CH₂)_(s),    (CH₂CH₂O)_(s′), (OCH₂CH₂CH₂)_(s″), (CH₂CH₂CH₂O)_(t), and (aa)_(t′);-   aa is independently at each occurrence an amino acid;-   Z is selected from the group: aryl substituted with 0-1 R¹⁶, C₃₋₁₀    cycloalkyl substituted with 0-1 R¹⁶, and a 5-10 membered    heterocyclic ring system containing 1-4 heteroatoms independently    selected from N, S, and O and substituted with 0-1 R¹⁶;-   R¹³, R^(13a), R¹⁴, R^(14a), and R¹⁵ are independently selected at    each occurrence from the group: H, ═O, COOH, SO₃H, C₁-C₅ alkyl    substituted with 0-1 R¹⁶, aryl substituted with 0-1 R¹⁶, benzyl    substituted with 0-1 R¹⁶, and C₁-C₅ alkoxy substituted with 0-1 R¹⁶,    NHC(═O)R¹⁷, C(═O)NHR¹⁷, NHC(═O)NHR¹⁷, NHR¹⁷, R¹⁷, and a bond to the    surfactant;-   k is 0 or 1;-   s is selected from 0, 1, 2, 3, 4, and 5;-   s′ is selected from 0, 1, 2, 3, 4, and 5;-   s″ is selected from 0, 1, 2, 3, 4, and 5; and-   t is selected from 0, 1, 2, 3, 4, and 5.

(62) A diagnostic agent according to embodiment 60 wherein:

-   W¹ is C(═O)NR¹⁵;-   h is 1;-   g is 3;-   R¹³ and R¹⁴ are independently H;-   x is 1;-   k is 0;-   g′ is 0;-   h′ is 1;-   W² is NH; and-   x′ is 1.

(63) A diagnostic agent according to embodiment 60

-   x is 0;-   k is 1;-   Z is aryl substituted with 0-3 R¹⁶;-   g′ is 1;-   W² is NH;-   R^(13a) and R^(14a) are independently H;-   h′ is 1; and-   x′ is 1.

(64) A diagnostic agent according to embodiment 60

-   W¹ is C(═O)NR¹⁵;-   h is 1;-   g is 2;-   R¹³ and R¹⁴ are independently H;-   x is 1;-   k is 0;-   g′ is 1;-   R^(13a) and R^(14a) are independently H; or C1-5 alkyl substituted    with 0-3 R¹⁶;-   R¹⁶ is SO₃H;-   W² is NHC(═O) or NH;-   h′ is 1; and-   x′ is 2.

(65) A diagnostic agent according to embodiment 60

-   W¹ is C(═O)NH;-   h is 1;-   g is 3;-   R¹³ and R¹⁴ are independently H;-   k is 0;-   g′ is 0;-   x is 1;-   W² is —NH(C═O)— or —(OCH₂CH₂)₇₆₋₈₄—;-   h′ is 2; and-   x′ is 1.

(66) A diagnostic agent according to embodiment 60

-   x is 0;-   k is 0;-   g′ is 3;-   h′ is 1;-   W² is NH; and-   x′ is 1.

(67) A diagnostic agent according to embodiment 60

-   x is 0;-   Z is aryl substituted with 0-3 R¹⁶;-   k is 1;-   g′ is 1;-   R^(13a)R^(14a) are independently H;-   W² is NHC(═O) or —(OCH₂CH₂)₇₆₋₈₄—; and-   x′ is 1.

(68) A diagnostic agent according to embodiment 60

-   W¹ is C═O;-   g is 2;-   R¹³ and R¹⁴ are independently H;-   k is 0;-   g′ is 0;-   h′ is 1;-   W² is NH; and-   x′ is 1.

(69) A diagnostic agent according to embodiment 48 wherein the linkinggroup is present.

(70) A diagnostic agent according to any one of embodiments 48-69wherein

-   S_(f) is a surfactant which is a lipid or a compound of the formula:-   A⁹ is selected from the group: OH and OR³²;-   A¹⁰ is OR³²;-   R³² is C(═O)C₁₋₂₀ alkyl;-   E⁹ is C₁₋₁₀ alkylene substituted with 1-3 R³³;-   R³³ is independently selected at each occurrence from the group:    R³⁵, —PO₃H—R³⁵, ═O, —CO₂R³⁴, —C(═O)R³⁴, —C(═O)N(R³⁴)₂, —CH₂OR³⁴,    —OR³⁴, —N(R³⁴)₂, C₁-C₅ alkyl, and C₂-C₄ alkenyl;-   R³⁴ is independently selected at each occurrence from the group:    R³⁵, H, C₁-C₆ alkyl, phenyl, benzyl, and trifluoromethyl;-   R³⁵ is a bond to L_(n);-   and a pharmaceutically acceptable salt thereof.

(71) A diagnostic agent according to any one of embodiments 48-70wherein the surfactant is a lipid or a compound of the formula:

-   A⁹ is OR³²,-   A¹⁰ is OR³²;-   R³² is C(═O)C₁₋₁₅ alkyl;-   E⁹ is C₁₋₄ alkylene substituted with 1-3 R³³;    wherein:-   A¹ ia a bond to Ln;-   E¹ is C₁, alkyl substituted by R²³;-   A² is NH;-   E² is C₂ alkyl sunsttuted wth 0-1R²³;-   A³ is —O—P(O)(R²¹)—O;-   E³ is C₁, alkyl;-   A⁴ and A⁵ are each —O—;-   E⁴ and E⁶ are each independently C₁₋₁₆ alkyl substituted with    0-1R²³;-   E⁵ is C₁, alkyl;-   A⁵ is —O—;-   R²¹ is —OH; and-   R²³ is ═O.

(73) A diagnostic agent according to embodiment 48 wherein the compoundis of the formula:(Q)_(d)-L_(n)-S_(f)

-   wherein, Q is a compound of Formulae (Ia) or (Ib):-   R is independently OH or —CH₂SH;

(73) A diagnostic agent according to embodiment 48 wherein the compoundis of the formula:(Q)_(d)-L_(n)-S_(f)

-   wherein, Q is a compound of Formulae (Ia) or (Ib):-   R is independently OH or —CH₂SH;-   R¹ is independently selected at each occurrence from the group: H,    OH, C₁₋₃ alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl, and heterocycle-S—CH₂—;-   R² is independently C₁₋₂₀ alkyl;-   X is independently C═O or SO₂, provided when X is C═O, R³ is    and when X is SO₂, R³ is independently selected from the group: aryl    substituted with 0-2 R⁶, and heterocycle substituted with 0-2 R⁶;-   R⁴ is independently selected at each occurrence from the group: C₁₋₆    alkyl, phenyl, and benzyl;-   R⁵ is independently at each occurrence from the group: NH(C₁₋₆    alkyl), NH-phenyl, and NH-heterocycle; wherein said alkyl, phenyl    and heterocycle groups are optionally substituted with a bond to    L_(n);-   R⁶ is independently aryloxy substituted with 0-3 R⁷;-   R⁷ is independently halogen or methoxy;-   or alternatively,-   R¹ and R⁴ may be taken together to form a bridging group of the    formula —(CH₂)₃—O-phenyl-CH₂—, optionally substituted with a bond to    L_(n);-   or alternatively,-   R¹ and R² may be taken together to form a bridging group of the    formula —(CH₂)₃—NH—, optionally substituted with a bond to L_(n); or-   R¹ and R² taken together with the nitrogen and carbon atom through    which they are attached form a C₅₋₇ atom saturated ring system    substituted with one or more substituents selected from the group    consisting of: a bond to Ln, a bond to Sf, and —C(═O)—NR²⁹R³⁰;-   R⁸ is independently at each occurrence OH or phenyl, optionally    substituted with a bond to L_(n), provided that when R⁸ is phenyl,    R¹⁰ is —C(═O)—CR¹²—NH—CH(CH₃)—COOH;-   R⁹ and R⁹′ are independently H, C₁₋₆ alkyl optionally substituted    with a bond to L_(n), or are taken together with the carbon atom to    which they are attached to form a 5-7 atom saturated, partially    unsaturated or aromatic ring system containing 0-3 heteroatoms    selected from O, N, SO₂ and S, said ring system substituted with R⁶    and optionally substituted with a bond to L_(n);-   R¹⁰ and R¹¹ are independently H, or C₁₋₆ alkyl optionally    substituted with a bond to L_(n), or are taken together with the    nitrogen atom to which they are attached to form a 5-7 atom    saturated, partially unsaturated or aromatic ring system containing    0-3 heteroatoms selected from O, N, SO₂ and S, said ring system    optionally substituted with 0-3 R²⁷ or a bond to L_(n);-   or alternatively,-   R⁹ and R¹⁰ are taken together with the carbon atom to which they are    attached to form a 5-7 atom saturated, partially unsaturated or    aromatic ring system containing 0-3 heteroatoms selected from O, N,    SO₂ and S, said ring system optionally substituted with a bond to    L_(n);-   R¹² is independently C₁₋₂₀ alkyl;-   d is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   L_(n) is a linking group having the formula:    ((W¹)_(h)—(CR¹³R¹⁴)_(g))_(x)-(Z)_(k)-((CR^(13a)R^(14a))_(g′)—(W²)_(h′))_(x′);-   W¹ and W² are independently selected at each occurrence from the    group: O, S, NH, NHC(═O), C(═O)NH, NR¹⁵C(═O), C(═O)NR¹⁵, C(═O),    C(═O)O, OC(═O), NHC(═S)NH, NHC(═O)NH, SO₂, SO₂NH, —(OCH₂CH₂)₇₆₋₈₄,    (OCH₂CH₂)_(s), (CH₂CH₂O)_(s′), (OCH₂CH₂CH₂)_(s″), (CH₂CH₂CH₂O)_(t),    and (aa)_(t′);-   aa is independently at each occurrence an amino acid;-   Z is selected from the group: aryl substituted with 0-3 R¹⁶, C₃₋₁₀    cycloalkyl substituted with 0-3 R¹⁶, and a 5-10 membered    heterocyclic ring system containing 1-4 heteroatoms independently    selected from N, S, and O and substituted with 0-3 R¹⁶;-   R¹³, R^(13a), R¹⁴, R^(14a), and R¹⁵ are independently selected at    each occurrence from the group: H, ═O, COOH, SO₃H, PO₃H, C₁-C₅ alkyl    substituted with 0-3 R¹⁶, aryl substituted with 0-3 R¹⁶, benzyl    substituted with 0-3 R¹⁶, and C₁-C₅ alkoxy substituted with 0-3 R¹⁶;    NHC(═O)R¹⁷, C(═O)NHR¹⁷, NHC(═O)NHR¹⁷, NHR¹⁷, R¹⁷, and a bond to Sf;-   R¹⁶ is independently selected at each occurrence from the group: a    bond to Sf, COOR¹⁷, C(═O)NHR¹⁷, NHC(═O)R¹⁷, OH, NHR¹⁷, SO₃H, PO₃H,    —OPO₃H₂, —OSO₃H, aryl substituted with 0-3 R¹⁷, C₁₋₅ alkyl    substituted with 0-1 R¹⁸, C₁₋₅ alkoxy substituted with 0-1 R¹⁸, and    a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms    independently selected from N, S, and O and substituted with 0-3    R¹⁷;-   R¹⁷ is independently selected at each occurrence from the group: H,    alkyl substituted with 0-1 R¹⁸, aryl substituted with 0-1 R¹⁸, a    5-10 membered heterocyclic ring system containing 1-4 heteroatoms    independently selected from N, S, and O and substituted with 0-1    R¹⁸, C₃₋₁₀ cycloalkyl substituted with 0-1 R¹⁸, polyalkylene glycol    substituted with 0-1 R¹⁸, carbohydrate substituted with 0-1 R¹⁸,    cyclodextrin substituted with 0-1 R¹⁸, amino acid substituted with    0-1 R¹⁸, polycarboxyalkyl substituted with 0-1 R¹⁸, polyazaalkyl    substituted with 0-1 R¹⁸, peptide substituted with 0-1 R¹⁸, wherein    the peptide is comprised of 2-10 amino acids,    3,6-O-disulfo-B-D-galactopyranosyl, bis(phosphonomethyl)glycine, and    a bond to Sf;-   R¹⁸ is a bond to Sf;-   k is selected from 0, 1, and 2;-   h is selected from 0, 1, and 2;-   h′ is selected from 0, 1, and 2;-   g is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   g′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   s is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   s′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   s″ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   t is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   t′ is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;-   x is selected from 0, 1, 2, 3, 4, and 5;-   x′ is selected from 0, 1, 2, 3, 4, and 5;-   S_(f) is a surfactant which is a lipid or a compound of the formula:-   A⁹ is selected from the group: OH and OR³²;-   A¹⁰ is OR³²;-   R³² is C(═O)C₁₋₂₀ alkyl;-   E⁹ is C₁₋₁₀ alkylene substituted with 1-3 R³³;-   R³³ is independently selected at each occurrence from the group:    R³⁵, —PO₃H—R³⁵, ═O, —CO₂R³⁴, —C(═O)R³⁴, —C(═O)N(R³⁴)₂, —CH₂OR³⁴,    —OR³⁴, —N(R³⁴)₂, C₁-C₅ alkyl, and C₂-C₄ alkenyl;-   R³⁴ is independently selected at each occurrence from the group:    R³⁵, H, C₁-C₆ alkyl, phenyl, benzyl, and trifluoromethyl;-   R³⁵ is a bond to L_(n); or-   Sf is of the formula:    wherein:-   A¹ ia a bond to Ln;-   E¹ is C₁ alkyl substituted by R²³;-   A² is NH;-   E² is C₂ alkyl sunsttuted wth 0-1R²³;-   A³ is —O—P(O)(R²¹)—O;-   E³ is C₁ alkyl;-   A⁴ and A⁵ are each —O—;-   E⁴ and E⁶ are each independently C₁₋₁₆ alkyl substituted with    0-1R²³;-   E⁵ is C₁ alkyl;-   A⁵ is —O—;-   R²¹ is —OH; and-   R²³ is ═O; or    -   a pharmaceutically acceptable salt thereof.

(74) A diagnostic agent according to embodiment 73, wherein:

-   R is —OH;-   R² is C₁₋₆ alkyl;-   X is C═O;-   R³ is-   R¹ and R⁴ are taken together to form a bridging group of formula    —(CH₂)₃—O-phenyl-CH₂—;-   R⁵ is NH(C1-6alkyl), substituted with a bond to the linking group or    a bond to the surfactant.

(75) A diagnostic agent according to any one of embodiments 73-74,wherein:

-   R is —OH;-   R⁹ is C₁ alkyl substituted with a bond to Ln;-   R¹⁰ and R¹¹ taken together with the nitrogen atom to which they are    attached form a 5 atom saturated ring system, said right system is    substituted with 0-3 R²⁷;-   R²⁷ is ═O, C1-4 alkyl, or phenyl substituted with R²⁸; and-   R²⁸ is a phenoxy group substituted with 0-2 OCH₃ groups;-   S_(f) is a surfactant which is a lipid or a compound of the formula:-   A⁹ is OR³²;-   A¹⁰ is OR³²;-   R³² is C(═O)C₁₋₁₅ alkyl;-   E⁹ is C₁₋₄ alkylene substituted with 1-3 R³³;-   R³³ is independently selected at each occurrence from the group:    R³⁵, —PO₃H—R³⁵, ═O, —CO₂R³⁴, —C(═O)R³⁴, —CH₂OR³⁴, —OR³⁴, and C₁-C₅    alkyl;-   R³⁴ is independently selected at each occurrence from the group:    R³⁵, H, C₁-C₆ alkyl, phenyl, and benzyl; and-   R³⁵ is a bond to L_(n).

(76) A diagnostic agent according according to any one of embodiments73-75, wherein:

-   R is —OH;-   R⁹ is C₁ alkyl substituted with a bond to Ln;-   R¹⁰ and R¹¹ taken together with the nitrogen atom to which they are    attached form a 5 atom saturated ring system, said right system is    substituted with 0-3 R²⁷;-   R²⁷ is ═O, C1-4 alkyl, or phenyl substituted with R²⁸; and-   R²⁸ is a phenoxy group substituted with 0-2 OCH₃ groups;-   S_(f) is a surfactant which is a lipid or a compound of the of the    formula:    wherein:-   A¹ ia a bond to Ln;-   E¹ is C₁ alkyl substituted by R²³;-   A² is NH;-   E² is C₂ alkyl sunsttuted wth 0-1R²³;-   A³ is —O—P(O)(R²¹)—O;-   E³ is C₁, alkyl;-   A⁴ and A⁵ are each —O—;-   E⁴ and E⁶ are each independently C₁₋₁₆ alkyl substituted with    0-1R²³;-   E⁵ is C₁ alkyl;-   A⁵ is —O—;-   R²¹ is —OH; and-   R²³ is ═O.

(77) A diagnostic agent according according to any one of embodiments73-76, wherein:

wherein

-   R is —OH;-   R¹ and R² taken together with the nitrogen and carbon atom through    which they are attached form a C₅₋₇ atom saturated ring system    substituted with one or more substituents selected from the group    consisting of: a bond to Ln, a bond to Sf, and —C(═O)—NR²⁹R³⁰;-   R²⁹ and R³⁰ taken together with the nitrogen atom through which they    are attached form a C5-7 atom saturated ring system substituted with    R³¹; and-   R³¹ is a benzyloxy group substituted with C1-4 alkyl.-   d is selected from 1, 2, 3, 4, and 5;-   W is independently selected at each occurrence from the group: O,    NH, NHC(═O), C(═O)NH, NR¹⁵C(═O), C(═O)NR¹⁵, C(═O),

C(═O)O, OC(═O), NHC(═S)NH, NHC(═O)NH, SO₂, (OCH₂CH₂)_(s),(CH₂CH₂O)_(s′), (OCH₂CH₂CH₂)_(s″), (CH₂CH₂CH₂O)_(t), and (aa)_(t′);

-   aa is independently at each occurrence an amino acid;-   Z is selected from the group: aryl substituted with 0-1 R¹⁶, C₃₋₁₀    cycloalkyl substituted with 0-1 R¹⁶, and a 5-10 membered    heterocyclic ring system containing 1-4 heteroatoms independently    selected from N, S, and O and substituted with 0-1 R¹⁶;-   R¹³, R^(13a), R¹⁴, R^(14a), and R¹⁵ are independently selected at    each occurrence from the group: H, ═O, COOH, SO₃H, C₁-C₅ alkyl    substituted with 0-1 R¹⁶, aryl substituted with 0-1 R¹⁶, benzyl    substituted with 0-1 R¹⁶, and C₁-C₅ alkoxy substituted with 0-1 R¹⁶,    NHC(═O)R¹⁷, C(═O)NHR¹⁷, NHC(═O)NHR¹⁷, NHR¹⁷, R¹⁷, and a bond to Sf;-   k is 0 or 1;-   s is selected from 0, 1, 2, 3, 4, and 5;-   s′ is selected from 0, 1, 2, 3, 4, and 5;-   s″ is selected from 0, 1, 2, 3, 4, and 5; and-   t is selected from o, 1, 2, 3, 4, and 5.

(78) A diagnostic agent according to according to any one of embodiments73-77, wherein:

-   W¹ is C(═O)NR¹⁵;-   h is 1;-   g is 3;-   R¹³ and R¹⁴ are independently H;-   x is 1;-   k is 0;-   g′ is O;-   h′ is 1;-   W² is NH; and-   x′ is 1.

(79) A diagnostic agent according to embodiment 73, wherein:

-   x is 0;-   k is 1;-   Z is aryl substituted with 0-3 R¹⁶;-   g′ is 1;-   W² is NH;-   R^(13a) and R^(14a) are independently H;-   h′ is 1; and-   x′ is 1.

(80) A diagnostic agent according to Embodiment 73, wherein:

-   W¹ is C(═O)NR¹⁵;-   h is 1;-   g is 2;-   R¹³ and R¹⁴ are independently H;-   x is 1;-   k is 0;-   g′ is 1;-   R^(13a) and R^(14a) are independently H; or C₁₋₅ alkyl substituted    with 0-3 R¹⁶;-   R¹⁶ is SO₃H;-   W² is NHC(═O) or NH;-   h′ is 1; and-   x′ is 2.

(81) A diagnostic agent according to Embodiment 73, wherein:

-   W¹ is C(═O)NH;-   h is 1;-   g is 3;-   R¹³ and R¹⁴ are independently H;-   k is 0;-   g′ is 0;-   x is 1;-   W² is —NH(C═O)— or —(OCH₂CH₂)₇₆₋₈₄—;-   h′ is 2; and-   x′ is 1.

(82) A diagnostic agent according to Embodiment 73, wherein:

-   x is 0;-   k is 0;-   g′ is 3;-   h′ is 1;-   W² is NH; and-   x′ is 1.

(83) A diagnostic agent according to Embodiment 73, wherein:

-   x is 0;-   Z is aryl substituted with 0-3 R¹⁶;-   k is 1;-   g′ is 1;-   R^(13a)R^(14a) are independently H;-   W² is NHC(═O) or —(OCH₂CH₂)₇₆₋₈₄—; and-   x′ is 1.

(84) A diagnostic agent according to Embodiment 73, wherein:

-   W¹ is C═O;-   g is 2;-   R¹³ and R¹⁴ are independently H;-   k is 0;-   g′ is 0;-   h′ is 1;-   W² is NH; and-   x′ is 1.

(85) A diagnostic agent according to Embodiment 1, wherein the compoundis selected from the group consisting of:

(86) A diagnostic agent according to embodiment 48, wherein: wherein theechogenic gas is a perfluorocarbon gas or sulfur hexafluoride.

(87) A diagnostic agent according to embodiment 86 wherein saidperfluorocarbon is selected from the group consisting ofperfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane,perfluorocyclobutane, perfluoropentane, and perfluorohexane.

(88) A diagnostic composition comprising a compound according toembodiment 48 or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier.

(89) A diagnostic composition comprising a compound according toembodiment 48 or a pharmaceutically acceptable salt form thereof, anechogenic gas and a pharmaceutically acceptable carrier.

(90) A diagnostic composition comprising a compound according toembodiment 48 further comprising:1,2-dipalmitoyl-sn-glycero-3-phosphotidic acid,1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, andN-(methoxypolyethylene glycol 5000carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine.

(91) A method of detecting, imaging or monitoring the presence of matrixmetalloproteinase in a patient comprising the steps of:

-   -   a) administering to said patient a diagnostic agent of        embodiment 1; and    -   b) acquiring an image of a site of concentration of said        diagnostic agent in the patient by a diagnostic imaging        technique.

(92) A method of detecting, imaging or monitoring the presence of matrixmetalloproteinase in a patient comprising the steps of:

-   -   a) administering to said patient a diagnostic agent of        embodiment 48; and    -   b) acquiring an image of a site of concentration of said        diagnostic agent in the patient by a diagnostic imaging        technique.

(93) A method of detecting, imaging or monitoring a pathologicaldisorder associated with matrix metalloproteinase activity in a patientcomprising the steps of:

-   -   a) administering to said patient a diagnostic agent of        embodiment 1; and    -   b) acquiring an image of a site of concentration of said        diagnostic agent in the patient by a diagnostic imaging        technique.

(94) A method of detecting, imaging or monitoring a pathologicaldisorder associated with matrix metalloproteinase activity in a patientcomprising the steps of:

-   -   a) administering to said patient a diagnostic agent according to        embodiment 48; and    -   b) acquiring an image of a site of concentration of said        diagnostic agent in the patient by a diagnostic imaging        technique.

(95) A method of detecting, imaging or monitoring atherosclerosis in apatient comprising the steps of:

-   -   a) administering a diagnostic agent according to embodiment 1;        and    -   b) acquiring an image of a site of concentration of said        diagnostic agent in the body by a diagnostic imaging technique.

(96) A method of detecting, imaging or monitoring atherosclerosis in apatient comprising the steps of:

-   -   a) administering a diagnostic agent according to embodiment 48;        and    -   b) acquiring an image of a site of concentration of said        diagnostic agent in the body by a diagnostic imaging technique.

(97) A method according to embodiment 95, wherein the atherosclerosis iscoronory atherosclerosis or cerebrovascular atherosclerosis.

(98) A method according to embodiment 96, wherein the atherosclerosis iscoronory atherosclerosis or cerebrovascular atherosclerosis.

(99) A method of identifying a patient at high risk for transientischemic attacks or stroke by determining the degree of activeatherosclerosis in a patient comprising carrying out the method ofembodiment 96.

(100) A method of identifying a patient at high risk for transientischemic attacks or stroke by determining the degree of activeatherosclerosis in a patient comprising carrying out the method ofembodiment 97.

(101) A method of identifying a patient at high risk for acute cardiacischemia, myocardial infarction or cardiac death by determining thedegree of active atherosclerosis by imaging the patient by the method ofembodiment 96.

(102) A method of identifying a patient at high risk for acute cardiacischemia, myocardial infarction or cardiac death by determining thedegree of active atherosclerosis by imaging the patient by the method ofembodiment 97.

(103) A method of simultaneous imaging of cardiac perfusion andextracellular matrix degradation in a patient comprising the steps of:

-   -   a) administering a diagnostic agent according to embodiment 1,        wherein the diagnostic metal is a gamma-emitting radioisotope;        and    -   (b) administering a cardiac perfusion compound, wherein the        compound is radiolabeled with a gamma-emitting radioisotope        which exhibits a gamma emission energy that is spectrally        separable from the gamma emission energy of the diagnostic metal        conjugated to the targeting moiety in step (a); and    -   (c) acquiring, by a diagnostic imaging technique, simultaneous        images of the sites of concentration of the spectrally separable        gamma-emission energies of the compounds administered in        steps (a) and (b).

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof.

Definitions

The compounds herein described may have asymmetric centers. Unlessotherwise indicated, all chiral, diastereomeric and racemic forms areincluded in the present invention. Many geometric isomers of olefins,C═N double bonds, and the like can also be present in the compoundsdescribed herein, and all such stable isomers are contemplated in thepresent invention. It will be appreciated that compounds of the presentinvention contain asymmetrically substituted carbon atoms, and may beisolated in optically active or racemic forms. It is well known in theart how to prepare optically active forms, such as by resolution ofracemic forms or by synthesis from optically active starting materials.Two distinct isomers (cis and trans) of the peptide bond are known tooccur; both can also be present in the compounds described herein, andall such stable isomers are contemplated in the present invention. The Dand L-isomers of a particular amino acid are designated herein using theconventional 3-letter abbreviation of the amino acid, as indicated bythe following examples: D-Leu, or L-Leu.

When any variable occurs more than one time in any substituent or in anyformula, its definition on each occurrence is independent of itsdefinition at every other occurrence. Thus, for example, if a group isshown to be substituted with 0-2 R⁵², then said group may optionally besubstituted with up to two R⁵², and R⁵² at each occurrence is selectedindependently from the defined list of possible R⁵². Also, by way ofexample, for the group —N(R⁵³)₂, each of the two R⁵³ substituents on Nis independently selected from the defined list of possible R⁵³.Combinations of substituents and/or variables are permissible only ifsuch combinations result in stable compounds. When a bond to asubstituent is shown to cross the bond connecting two atoms in a ring,then such substituent may be bonded to any atom on the ring.

The term “metallopharmaceutical” means a pharmaceutical comprising ametal. The metal is the cause of the imageable signal in diagnosticapplications and the source of the cytotoxic radiation inradiotherapeutic applications. Radiopharmaceuticals aremetallopharmaceuticals in which the metal is a radioisotope.

By “reagent” is meant a compound of this invention capable of directtransformation into a metallopharmaceutical of this invention. Reagentsmay be utilized directly for the preparation of themetallopharmaceuticals of this invention or may be a component in a kitof this invention.

The term “inhibitor” means a compound of this invention that inhibitsthe function of a matrix metalloproteinase.

By “stable compound” or “stable structure” is meant herein a compoundthat is sufficiently robust to survive isolation to a useful degree ofpurity from a reaction mixture, and formulation into an efficaciouspharmaceutical agent.

The term “substituted”, as used herein, means that one or more hydrogenson the designated atom or group is replaced with a selection from theindicated group, provided that the designated atom's or group's normalvalency is not exceeded, and that the substitution results in a stablecompound. When a substituent is keto (i.e., ═O), then 2 hydrogens on theatom are replaced.

The term “bond”, as used herein, means either a single or double bond.

The term “salt”, as used herein, is used as defined in the CRC Handbookof Chemistry and Physics, 65th Edition, CRC Press, Boca Raton, Fla.,1984, as any substance which yields ions, other than hydrogen orhydroxyl ions. As used herein, “pharmaceutically acceptable salts” referto derivatives of the disclosed compounds modified by making acid orbase salts. Examples of pharmaceutically acceptable salts include, butare not limited to, mineral or organic acid salts of basic residues suchas amines; alkali or organic salts of acidic residues such as carboxylicacids; and the like.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the disclosed compounds wherein the parent compound is modified bymaking acid or base salts thereof. Examples of pharmaceuticallyacceptable salts include, but are not limited to, mineral or organicacid salts of basic residues such as amines; alkali or organic salts ofacidic residues such as carboxylic acids; and the like. Thepharmaceutically acceptable salts include the conventional non-toxicsalts or the quaternary ammonium salts of the parent compound formed,for example, from non-toxic inorganic or organic acids. For example,such conventional non-toxic salts include those derived from inorganicacids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric,nitric and the like; and the salts prepared from organic acids such asacetic, propionic, succinic, glycolic, stearic, lactic, tartaric,citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,and the like.

The pharmaceutically acceptable salts of the present invention can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, p. 1418, the disclosure of which is hereby incorporated byreference.

As used herein, “alkyl” is intended to include both branched andstraight-chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms, examples of which include, but are notlimited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl,sec-butyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl;“cycloalkyl” or “carbocycle” is intended to include saturated andpartially unsaturated ring groups, including mono-, bi- or poly-cyclicring systems, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl and adamantyl; “bicycloalkyl” or “bicyclic” isintended to include saturated bicyclic ring groups such as[3.3.0]bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane(decalin), [2.2.2]bicyclooctane, and so forth.

As used herein, the term “alkene” or “alkenyl”, is intended to includehydrocarbon chains having the specified number of carbon atoms of eithera straight or branched configuration and one or more unsaturatedcarbon-carbon bonds which may occur in any stable point along the chain,such as ethenyl, propenyl, and the like.

As used herein, the term “alkyne” or “alkynyl” is intended to includehydrocarbon chains having the specified number of carbon atoms of eithera straight or branched configuration and one or more unsaturatedcarbon-carbon triple bonds which may occur in any stable point along thechain, such as propargyl, and the like.

As used herein, “aryl” or “aromatic residue” is intended to mean phenylor naphthyl, which when substituted, the substitution can be at anyposition.

As used herein, the term “heterocycle” or “heterocyclic system” isintended to mean a stable 5- to 7-membered monocyclic or bicyclic or 7-to 10-membered bicyclic heterocyclic ring which is saturated partiallyunsaturated or unsaturated (aromatic), and which consists of carbonatoms and from 1 to 4 heteroatoms independently selected from the groupconsisting of N, O and S and including any bicyclic group in which anyof the above-defined heterocyclic rings is fused to a benzene ring. Thenitrogen and sulfur heteroatoms may optionally be oxidized. Theheterocyclic ring may be attached to its pendant group at any heteroatomor carbon atom which results in a stable structure. The heterocyclicrings described herein may be substituted on carbon or on a nitrogenatom if the resulting compound is stable. If specifically noted, anitrogen in the heterocycle may optionally be quaternized. It ispreferred that when the total number of S and O atoms in the heterocycleexceeds 1, then these heteroatoms are not adjacent to one another. It ispreferred that the total number of S and O atoms in the heterocycle isnot more than 1. As used herein, the term “aromatic heterocyclic system”is intended to mean a stable 5- to 7-membered monocyclic or bicyclic or7- to 10-membered bicyclic heterocyclic aromatic ring which consists ofcarbon atoms and from 1 to 4 heteroatoms independently selected from thegroup consisting of N, O and S. It is preferred that the total number ofS and O atoms in the aromatic heterocycle is not more than 1.

Examples of heterocycles include, but are not limited to, 1H-indazole,2-pyrrolidonyl, 2H,6H-1,5,2-dithiazinyl, 2H-pyrrolyl, 3H-indolyl,4-piperidonyl, 4aH-carbazole, 4H-quinolizinyl, 6H-1,2,5-thiadiazinyl,acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl,benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazalonyl,carbazolyl, 4aH-carbazolyl, □-carbolinyl, chromanyl, chromenyl,cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, isobenzofuranyl, isochromanyl, isoindazolyl,isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl,1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl,1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinylperimidinyl,phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl,phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl,piperidinyl, pteridinyl, piperidonyl, 4-piperidonyl, pteridinyl,purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl,pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl,pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, quinazolinyl,quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, carbolinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl,1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl,thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl,triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl,1,3,4-triazolyl, xanthenyl. Preferred heterocycles include, but are notlimited to, pyridinyl, furanyl, thienyl, pyrrolyl, pyrazolyl,imidazolyl, indolyl, benzimidazolyl, 1H-indazolyl, oxazolidinyl,benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, or isatinoyl.Also included are fused ring and spiro compounds containing, forexample, the above heterocycles.

As used herein, the term “alkaryl” means an aryl group bearing an alkylgroup of 1-10 carbon atoms; the term “aralkyl”, means an alkyl group of1-10 carbon atoms bearing an aryl group; the term “arylalkaryl” means anaryl group bearing an alkyl group of 1-10 carbon atoms bearing an arylgroup; and the term “heterocycloalkyl” means an alkyl group of 1-10carbon atoms bearing a heterocycle.

A “polyalkylene glycol” is a polyethylene glycol, polypropylene glycolor polybutylene glycol having a molecular weight of less than about5000, terminating in either a hydroxy or alkyl ether moiety.

A “carbohydrate” is a polyhydroxy aldehyde, ketone, alcohol or acid, orderivatives thereof, including polymers thereof having polymericlinkages of the acetal type.

A “cyclodextrin” is a cyclic oligosaccharide. Examples of cyclodextrinsinclude, but are not limited to, □-cyclodextrin,hydroxyethyl-□-cyclodextrin, hydroxypropyl-□-cyclodextrin,□-cyclodextrin, hydroxypropyl-□-cyclodextrin,carboxymethyl-□-cyclodextrin, dihydroxypropyl-□-cyclodextrin,hydroxyethyl-□-cyclodextrin, 2,6 di-O-methyl-□-cyclodextrin,sulfated-□-cyclodextrin, □-cyclodextrin, hydroxypropyl-□-cyclodextrin,dihydroxypropyl-□-cyclodextrin, hydroxyethyl-□-cyclodextrin, andsulfated □-cyclodextrin.

As used herein, the term “polycarboxyalkyl” means an alkyl group havingbetween two and about 100 carbon atoms and a plurality of carboxylsubstituents; and the term “polyazaalkyl” means a linear or branchedalkyl group having between two and about 100 carbon atoms, interruptedby or substituted with a plurality of amine groups.

A “reducing agent” is a compound that reacts with a radionuclide, whichis typically obtained as a relatively unreactive, high oxidation statecompound, to lower its oxidation state by transferring electron(s) tothe radionuclide, thereby making it more reactive. Reducing agentsuseful in the preparation of radiopharmaceuticals and in diagnostic kitsuseful for the preparation of said radiopharmaceuticals include but arenot limited to stannous chloride, stannous fluoride, formamidinesulfinic acid, ascorbic acid, cysteine, phosphines, and cuprous orferrous salts. Other reducing agents are described in Brodack et. al.,PCT Application 94/22496, which is incorporated herein by reference.

A “transfer ligand” is a ligand that forms an intermediate complex witha metal ion that is stable enough to prevent unwanted side-reactions butlabile enough to be converted to a metallopharmaceutical. The formationof the intermediate complex is kinetically favored while the formationof the metallopharmaceutical is thermodynamically favored. Transferligands useful in the preparation of metallopharmaceuticals and indiagnostic kits useful for the preparation of diagnosticradiopharmaceuticals include but are not limited to gluconate,glucoheptonate, mannitol, glucarate,N,N,N′,N′-ethylenediaminetetraacetic acid, pyrophosphate andmethylenediphosphonate. In general, transfer ligands are comprised ofoxygen or nitrogen donor atoms.

The term “donor atom” refers to the atom directly attached to a metal bya chemical bond.

“Ancillary” or “co-ligands” are ligands that are incorporated into aradiopharmaceutical during its synthesis. They serve to complete thecoordination sphere of the radionuclide together with the chelator orradionuclide bonding unit of the reagent. For radiopharmaceuticalscomprised of a binary ligand system, the radionuclide coordinationsphere is composed of one or more chelators or bonding units from one ormore reagents and one or more ancillary or co-ligands, provided thatthere are a total of two types of ligands, chelators or bonding units.For example, a radiopharmaceutical comprised of one chelator or bondingunit from one reagent and two of the same ancillary or co-ligands and aradiopharmaceutical comprised of two chelators or bonding units from oneor two reagents and one ancillary or co-ligand are both considered to becomprised of binary ligand systems. For radiopharmaceuticals comprisedof a ternary ligand system, the radionuclide coordination sphere iscomposed of one or more chelators or bonding units from one or morereagents and one or more of two different types of ancillary orco-ligands, provided that there are a total of three types of ligands,chelators or bonding units. For example, a radiopharmaceutical comprisedof one chelator or bonding unit from one reagent and two differentancillary or co-ligands is considered to be comprised of a ternaryligand system.

Ancillary or co-ligands useful in the preparation ofradiopharmaceuticals and in diagnostic kits useful for the preparationof said radiopharmaceuticals are comprised of one or more oxygen,nitrogen, carbon, sulfur, phosphorus, arsenic, selenium, and telluriumdonor atoms. A ligand can be a transfer ligand in the synthesis of aradiopharmaceutical and also serve as an ancillary or co-ligand inanother radiopharmaceutical. Whether a ligand is termed a transfer orancillary or co-ligand depends on whether the ligand remains in theradionuclide coordination sphere in the radiopharmaceutical, which isdetermined by the coordination chemistry of the radionuclide and thechelator or bonding unit of the reagent or reagents.

A “chelator” or “bonding unit” is the moiety or group on a reagent thatbinds to a metal ion through the formation of chemical bonds with one ormore donor atoms.

The term “binding site” means the site in vivo or in vitro that binds abiologically active molecule.

A “diagnostic kit” or “kit” comprises a collection of components, termedthe formulation, in one or more vials which are used by the practicingend user in a clinical or pharmacy setting to synthesize diagnosticradiopharmaceuticals. The kit provides all the requisite components tosynthesize and use the diagnostic radiopharmaceutical except those thatare commonly available to the practicing end user, such as water orsaline for injection, a solution of the radionuclide, equipment forheating the kit during the synthesis of the radiopharmaceutical, ifrequired, equipment necessary for administering the radiopharmaceuticalto the patient such as syringes and shielding, and imaging equipment.

X-ray contrast agent pharmaceuticals, ultrasound contrast agentpharmaceuticals and metallopharmaceuticals for magnetic resonanceimaging contrast are provided to the end user in their final form in aformulation contained typically in one vial, as either a lyophilizedsolid or an aqueous solution. The end user reconstitutes the lyophilizedwith water or saline and withdraws the patient dose or just withdrawsthe dose from the aqueous solution formulation as provided.

A “lyophilization aid” is a component that has favorable physicalproperties for lyophilization, such as the glass transition temperature,and is added to the formulation to improve the physical properties ofthe combination of all the components of the formulation forlyophilization.

A “stabilization aid” is a component that is added to themetallopharmaceutical or to the diagnostic kit either to stabilize themetallopharmaceutical or to prolong the shelf-life of the kit before itmust be used. Stabilization aids can be antioxidants, reducing agents orradical scavengers and can provide improved stability by reactingpreferentially with species that degrade other components or themetallopharmaceutical.

A “solubilization aid” is a component that improves the solubility ofone or more other components in the medium required for the formulation.

A “bacteriostat” is a component that inhibits the growth of bacteria ina formulation either during its storage before use of after a diagnostickit is used to synthesize a radiopharmaceutical.

The following abbreviations are used herein:

-   Acm acetamidomethyl-   b-Ala, beta-Ala or bAla 3-aminopropionic acid-   ATA 2-aminothiazole-5-acetic acid or 2-aminothiazole-5-acetyl group-   Boc t-butyloxycarbonyl-   CBZ, Cbz or Z Carbobenzyloxy-   Cit citrulline-   Dap 2,3-diaminopropionic acid-   DCC dicyclohexylcarbodiimide-   DIEA diisopropylethylamine-   DMAP 4-dimethylaminopyridine-   EOE ethoxyethyl-   HBTU 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium    hexafluorophosphate-   hynic boc-hydrazinonicotinyl group or    2-[[[5-[carbonyl]-2-pyridinyl]hydrazono]methyl]-benzenesulfonic    acid,-   NMeArg or MeArg a-N-methyl arginine-   NMeAsp a-N-methyl aspartic acid-   NMM N-methylmorpholine-   OcHex O-cyclohexyl-   OBzl O-benzyl-   oSu O-succinimidyl-   TBTU 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium    tetrafluoroborate-   THF tetrahydrofuranyl-   THP tetrahydropyranyl-   Tos tosyl-   Tr trityl

The following conventional three-letter amino acid abbreviations areused herein; the conventional one-letter amino acid abbreviations areNOT used herein:

-   -   Ala=alanine    -   Arg=arginine    -   Asn=asparagine    -   Asp=aspartic acid    -   Cys=cysteine    -   Gln=glutamine    -   Glu=glutamic acid    -   Gly=glycine    -   His=histidine    -   Ile=isoleucine    -   Leu=leucine    -   Lys=lysine    -   Met=methionine    -   Nle=norleucine    -   Orn=ornithine    -   Phe=phenylalanine    -   Phg=phenylglycine    -   Pro=proline    -   Sar=sarcosine    -   Ser=serine    -   Thr=threonine    -   Trp=tryptophan    -   Tyr=tyrosine    -   Val=valine

The ultrasound contrast agents of the present invention comprise aplurality of matrix metalloproteinase inhibiting moieties attached to orincorporated into a microbubble of a biocompatible gas, a liquidcarrier, and a surfactant microsphere, further comprising an optionallinking moiety, L_(n), between the targeting moieties and themicrobubble. In this context, the term liquid carrier means aqueoussolution and the term surfactant means any amphiphilic material whichproduces a reduction in interfacial tension in a solution. A list ofsuitable surfactants for forming surfactant microspheres is disclosed inEP0727225A2, herein incorporated by reference. The term surfactantmicrosphere includes nanospheres, liposomes, vesicles and the like. Thebiocompatible gas can be air, or a fluorocarbon, such as a C₃-C₅perfluoroalkane, which provides the difference in echogenicity and thusthe contrast in ultrasound imaging. The gas is encapsulated or containedin the microsphere to which is attached the biodirecting group,optionally via a linking group. The attachment can be covalent, ionic orby van der Waals forces. Specific examples of such contrast agentsinclude lipid encapsulated perfluorocarbons with a plurality of MMPinhibiting compounds.

X-ray contrast agents of the present invention are comprised of one ormore matrix metalloproteinase inhibiting targeting moieties attached toone or more X-ray absorbing or “heavy” atoms of atomic number 20 orgreater, further comprising an optional linking moiety, L_(n), betweenthe targeting moieties and the X-ray absorbing atoms. The frequentlyused heavy atom in X-ray contrast agents is iodine. Recently, X-raycontrast agents comprised of metal chelates (Wallace, R., U.S. Pat. No.5,417,959) and polychelates comprised of a plurality of metal ions(Love, D., U.S. Pat. No. 5,679,810) have been disclosed. More recently,multinuclear cluster complexes have been disclosed as X-ray contrastagents (U.S. Pat. No. 5,804,161, PCT WO91/14460, and PCT WO 92/17215).

MRI contrast agents of the present invention are comprised of one ormore matrix metalloproteinase inhibiting targeting moieties attached toone or more paramagnetic metal ions, further comprising an optionallinking moiety, Ln, between the targeting moieties and the paramagneticmetal ions. The paramagnetic metal ions are present in the form of metalcomplexes or metal oxide particles. U.S. Pat. Nos. 5,412,148, and5,760,191, describe examples of chelators for paramagnetic metal ionsfor use in MRI contrast agents. U.S. Pat. No. 5,801,228, U.S. Pat. No.5,567,411, and U.S. Pat. No. 5,281,704, describe examples ofpolychelants useful for complexing more than one paramagnetic metal ionfor use in MRI contrast agents. U.S. Pat. No. 5,520,904, describesparticulate compositions comprised of paramagnetic metal ions for use asMRI contrast agents.

Matrix metalloproteinases (MMPs) are a family of structurally relatedzinc-containing enzymes that mediate the integrity of extracellularmatrix (Whittaker, M. et al, Chem. Rev., 1999, 99, 2735-2776). They areexcreted by a variety of connective tissue and pro-inflammatory cells,such as, fibroblasts, osteoblasts, macrophages, neutrophils, lymphocytesand endothelial cells. There is now a body of evidence that matrixmetalloproteinases (MMPs) are important in the uncontrolled breakdown ofconnective tissue, including proteoglycan and collagen, leading toresorption of the extracellular matrix. This is a feature of a number ofcardiovascular pathological conditions, such as atherosclerosis, heartfailure, restenosis and reperfusion injury. Normally these catabolicenzymes are tightly regulated at the level of their synthesis, as wellas, at their level of extracellular activity through the action ofspecific inhibitors, such as alpha-2-macroglobulins and TIMP (tissueinhibitor of metalloproteinase), which form inactive complexes with theMMPs. Therefore, extracellular matrix degradation and remodeling areregulated by the relative expression of TIMPs and MMPs. The MMPs areclassified into several families based on their domain structure:matrilysin (minimal domain, MMP-7), collagenase (hemopexin domain,MMP-1, MMP-8, MMP-13), gelatinase (fibronectin domain, MMP-2, MMP-9),stromelysin (hemopexin domain, MMP-3, MMP-10, MMP-11), metalloelastase(MMP-12). In addition, the transmembrane domain family (MT-MMPs) hasbeen recently discovered and comprises MMP-14 through MMP-17.

MMP proteolytic activity and extracellular matrix degradation isdependent on the comparative balance between MMPs and TIMPs. TIMPs andsynthetic small molecules or matrix metalloproteinase inhibitors havetherapeutic potential for diseases involving elevated levels of MMPactivity (Whittaker, M. et al, Chem. Rev., 1999, 99, 2735-2776; Babine,R. E. et al, Chem. Rev., 1997, 97, 1359; De, B. et al, Ann. N.Y. Acad.Sci., 1999, 878, 40-60; Summers, J. B. et al, Annual Reports in Med.Chem., 1998, 33, 131).

A functional group, such as —CONH—OH, —COOH, or —SH, is necessary for amolecule to be an effective inhibitor of MMPs. This functional group isinvolved in the chelation of the active site zinc ion, and is commonlyreferred to as the zinc binding group or ZBG. The hydroxamate, forexample, is a bidentate ligand for zinc.

The pharmaceuticals of the present invention have the formulae,(Q)_(d)-L_(n)-(C_(h)—X), (Q)_(d)-L_(n)-(C_(h)—X¹)_(d′),(Q)_(d)-L_(n)-(X²)_(d″), and (Q)_(d)-L_(n)-(X³), wherein Q represents acompound that inhibits a matrix metalloproteinase, d is 1-10, d′=1-100,L_(n) represents an optional linking group, C_(h) represents a metalchelator or bonding moiety, X represents a radioisotope, X¹ representsparamagnetic metal ion, X² represents a paramagnetic metal ion or heavyatom containing insoluble solid particle, d″ is 1-100, and X³ representsa surfactant microsphere of an echogenic gas.

One class of compounds of the present invention is comprised of one ormore inhibitors, Q, which are succinyl hydroxamates. A generic structureof succinyl hydroxamate is shown below (1).

The ethylene spacer between the ZBG (—CONH—OH) and the succinyl amide isessential for potent activity. Substitution at P₁ tends to conferbroad-spectrum activity on the MMPIs. Substituents at this position, ingeneral, tend to point away from the enzyme. Moieties capable ofhydrogen bonding and lipophilic substituents at the P₁ position α to thehydroxamate (Johnson, W. H. et al, J. Enz. Inhib., 1987, 2, 1) tend toenhance activity (2). Incorporation of a hydroxyl group (Beckett, P. R.,et al, Drug Discovery Today, 1996, 1, 16) at that position improves oralactivity in some case (3).

Substituents at the P₁′ position on the succinyl hydroxamates tend toimpart selectivity to the MMPIs. The S₁′ pocket is deep for MMP-2,MMP-3, MMP-8 and MMP-9 and occluded (short) for MMP-1 and MMP-7. A longalkyl substituent at the P₁′ position, for example, imparts selectivity(Miller, A. et al, Bioorg. Med. Chem. Lett., 1997, 7, 193) for MMP-2over MMP-1 and MMP-3 (4 and 5).

Substituents at the P₂′ position also point away from the enzyme. The P₁and the P₂′ positions can be linked (Xue, C-B. et al, J. Med. Chem.,1998, 41, 1745; Steinman, D. H. et al, Bioorg. Med. Chem. Lett., 1998,8, 2087) to form a macrocycle (6). Compounds such as (6) also exhibitnanomolar activity.

The nature of the macrocycle also imparts some selective inhibitionamong the MMPs. The P₂′ and the P₃′ positions may be cyclized to formlactams. The size of the lactam governs the selectivity.

The P₃′ position is a relatively open area in the succinyl hydroxamates,and a wide range of substitutents (for example (7)) may be introduced(Sheppard, G. S. et al, Bioorg. Med. Chem. Lett., 1998, 8, 3251) at thisposition. This position also offers the flexibility of attaching theoptional linker, L_(n), the chelator(s0, C_(h), for the imageablemoieties X and X¹, and the imageable moieties, X² and X³.

Other succinyl hydroxamates with modified P₂′ and P₃′ positions, such as(8) also have shown potent inhibition of MMPs. Those compounds andsyntheses of them are further described in the following patentapplications which are hereby incorporated by reference into this patentapplication: U.S. patent application Ser. Nos. 09/165,747, 08/743,439,09/134,484, 09/247,675, 09/335,086, 09/312,066, 09/311,168, 60/127,594,and 60/127,635.

Another class of compounds of the present invention is comprised of oneor more inhibitors, Q, which are sulfonamide hydroxamates, such as (9)and (10). Modification of the isopropyl substituent in (10) results indeep pocket MMP selectivity, for example MMP-2 vs MMP-1 (Santos, O. etal., J. Clin. Exp. Metastasis, 1997, 15, 499; MacPherson, L. J. et al,J. Med. Chem., 1997, 40, 2525).

Additional examples of inhibitors, Q, include the derivatized ‘alanine’hydroxamates, such as compounds (11) and (12), which show selectivityfor MMP-2 and MMP-9 over the other MMPs. Those compounds and synthesesof them are further described in the following patent applications whichare hereby incorporated by reference into this patent application: U.S.patent application Ser. Nos. 09/165,747, 08/743,439, 09/134,484,09/247,675, 09/335,086, 09/312,066, 09/311,168, 60/127,594, and60/127,635.

compound (13), which shows selectivity for MMP-2 and MMP-9, and in whichthe alpha position has a quaternary carbon and the molecule does notcontain any stereo centers (Lovejoy, B. et al., Nature Struct. Biol.,1999, 6, 217);

compound (14), which uses a carboxylic acid as the ZBG, and exhibitssignificant selectivity for MMP-2 (vs MMP-1), when X=butyl vs X=H(Sahoo, S. P. et al, Bioorg. Med. Chem. Lett., 1995, 5, 2441); and

succinyl thiols such as (15) (Levin, J. I. et al, Bioorg. Med. Chem.Lett., 1998, 8, 1163), in which the P₃′ position may be utilized toattach the optional linker, L_(n), the chelator(s), C_(h), for theimageable moieties X and X¹, and the imageable moieties, X² and X³.

Preferred pharmaceuticals of the present invention are comprised ofinhibitors, Q, which exhibit selectivity for MMP-1, MMP-2, MMP-3, MMP-9,or MMP-14 alone or in combination over the other MMPs. Examples ofpreferred moieties, Q, include compounds 4, 5, 6, 8, 9, 10, 11, 12, and13.

Most preferred are comprised of inhibitors, Q, which exhibit selectivityfor MMP-2, MMP-9, or MMP-14 alone or in combination over the other MMPs.Examples of the most preferred moieties, Q, include compounds 6, 8, 11,and 12.

The pharmaceuticals of the present invention can be synthesized byseveral approaches. One approach involves the synthesis of the targetingMMP inhibiting moiety, Q, and direct attachment of one or more moieties,Q, to one or more metal chelators or bonding moieties, C_(h), or to aparamagnetic metal ion or heavy atom containing solid particle, or to anechogenic gas microbubble. Another approach involves the attachment ofone or more moieties, Q, to the linking group, L_(n), which is thenattached to one or more metal chelators or bonding moieties, C_(h), orto a paramagnetic metal ion or heavy atom containing solid particle, orto an echogenic gas microbubble. Another approach, useful in thesynthesis of pharmaceuticals wherein d is 1, involves the synthesis ofthe moiety, Q-L_(n), together, by incorporating residue bearing L_(n)into the synthesis of the MMP inhibitor, Q. The resulting moiety,Q-L_(n), is then attached to one or more metal chelators or bondingmoieties, C_(h), or to a paramagnetic metal ion or heavy atom containingsolid particle, or to an echogenic gas microbubble. Another approachinvolves the synthesis of an inhibitor, Q, bearing a fragment of thelinking group, L_(n), one or more of which are then attached to theremainder of the linking group and then to one or more metal chelatorsor bonding moieties, C_(h), or to a paramagnetic metal ion or heavy atomcontaining solid particle, or to an echogenic gas microbubble.

The MMP inhibiting moieties, Q, optionally bearing a linking group,L_(n), or a fragment of the linking group, can be synthesized usingstandard synthetic methods known to those skilled in the art. Preferredmethods include but are not limited to those methods described below.

Generally, peptides, polypeptides and peptidomimetics are elongated bydeprotecting the alpha-amine of the C-terminal residue and coupling thenext suitably protected amino acid through a peptide linkage using themethods described. This deprotection and coupling procedure is repeateduntil the desired sequence is obtained. This coupling can be performedwith the constituent amino acids in a stepwise fashion, or condensationof fragments (two to several amino acids), or combination of bothprocesses, or by solid phase peptide synthesis according to the methodoriginally described by Merrifield, J. Am. Chem. Soc., 85, 2149-2154(1963), the disclosure of which is hereby incorporated by reference.

The peptides, polypeptides and peptidomimetics may also be synthesizedusing automated synthesizing equipment. In addition to the foregoing,procedures for peptide, polypeptide and peptidomimetic synthesis aredescribed in Stewart and Young, “Solid Phase Peptide Synthesis”, 2nd ed,Pierce Chemical Co., Rockford, Ill. (1984); Gross, Meienhofer,Udenfriend, Eds., “The Peptides: Analysis, Synthesis, Biology, Vol. 1,2, 3, 5, and 9, Academic Press, New York, (1980-1987); Bodanszky,“Peptide Chemistry: A Practical Textbook”, Springer-Verlag, New York(1988); and Bodanszky et al. “The Practice of Peptide Synthesis”Springer-Verlag, New York (1984), the disclosures of which are herebyincorporated by reference.

The coupling between two amino acid derivatives, an amino acid and apeptide, polypeptide or peptidomimetic, two peptide, polypeptide orpeptidomimetic fragments, or the cyclization of a peptide, polypeptideor peptidomimetic can be carried out using standard coupling proceduressuch as the azide method, mixed carbonic acid anhydride (isobutylchloroformate) method, carbodiimide (dicyclohexylcarbodiimide,diisopropylcarbodiimide, or water-soluble carbodiimides) method, activeester (p-nitrophenyl ester, N-hydroxysuccinic imido ester) method,Woodward reagent K method, carbonyldiimidazole method, phosphorusreagents such as BOP-Cl, or oxidation-reduction method. Some of thesemethods (especially the carbodiimide) can be enhanced by the addition of1-hydroxybenzotriazole. These coupling reactions may be performed ineither solution (liquid phase) or solid phase.

The functional groups of the constituent amino acids or amino acidmimetics must be protected during the coupling reactions to avoidundesired bonds being formed. The protecting groups that can be used arelisted in Greene, “Protective Groups in Organic Synthesis” John Wiley &Sons, New York (1981) and “The Peptides: Analysis, Synthesis, Biology,Vol. 3, Academic Press, New York (1981), the disclosure of which ishereby incorporated by reference.

The alpha-carboxyl group of the C-terminal residue is usually protectedby an ester that can be cleaved to give the carboxylic acid. Theseprotecting groups include: 1) alkyl esters such as methyl and t-butyl,2) aryl esters such as benzyl and substituted benzyl, or 3) esters whichcan be cleaved by mild base treatment or mild reductive means such astrichloroethyl and phenacyl esters. In the solid phase case, theC-terminal amino acid is attached to an insoluble carrier (usuallypolystyrene). These insoluble carriers contain a group which will reactwith the carboxyl group to form a bond which is stable to the elongationconditions but readily cleaved later. Examples of which are: oxime resin(DeGrado and Kaiser (1980) J. Org. Chem. 45, 1295-1300) chloro orbromomethyl resin, hydroxymethyl resin, and aminomethyl resin. Many ofthese resins are commercially available with the desired C-terminalamino acid already incorporated.

The alpha-amino group of each amino acid must be protected. Anyprotecting group known in the art can be used. Examples of these are: 1)acyl types such as formyl, trifluoroacetyl, phthalyl, andp-toluenesulfonyl; 2) aromatic carbamate types such as benzyloxycarbonyl(Cbz) and substituted benzyloxycarbonyls,1-(p-biphenyl)-1-methylethoxycarbonyl, and 9-fluorenylmethyloxycarbonyl(Fmoc); 3) aliphatic carbamate types such as tert-butyloxycarbonyl(Boc), ethoxycarbonyl, diisopropylmethoxycarbonyl, and allyloxycarbonyl;4) cyclic alkyl carbamate types such as cyclopentyloxycarbonyl andadamantyloxycarbonyl; 5) alkyl types such as triphenylmethyl and benzyl;6) trialkylsilane such as trimethylsilane; and 7) thiol containing typessuch as phenylthiocarbonyl and dithiasuccinoyl. The preferredalpha-amino protecting group is either Boc or Fmoc. Many amino acid oramino acid mimetic derivatives suitably protected for peptide synthesisare commercially available.

The alpha-amino protecting group is cleaved prior to the coupling of thenext amino acid. When the Boc group is used, the methods of choice aretrifluoroacetic acid, neat or in dichloromethane, or HCl in dioxane. Theresulting ammonium salt is then neutralized either prior to the couplingor in situ with basic solutions such as aqueous buffers, or tertiaryamines in dichloromethane or dimethylformamide. When the Fmoc group isused, the reagents of choice are piperidine or substituted piperidinesin dimethylformamide, but any secondary amine or aqueous basic solutionscan be used. The deprotection is carried out at a temperature between 0°C. and room temperature.

Any of the amino acids or amino acid mimetics bearing side chainfunctionalities must be protected during the preparation of the peptideusing any of the above-identified groups. Those skilled in the art willappreciate that the selection and use of appropriate protecting groupsfor these side chain functionalities will depend upon the amino acid oramino acid mimetic and presence of other protecting groups in thepeptide, polypeptide or peptidomimetic. The selection of such aprotecting group is important in that it must not be removed during thedeprotection and coupling of the alpha-amino group.

For example, when Boc is chosen for the alpha-amine protection thefollowing protecting groups are acceptable: p-toluenesulfonyl (tosyl)moieties and nitro for arginine; benzyloxycarbonyl, substitutedbenzyloxycarbonyls, tosyl or trifluoroacetyl for lysine; benzyl or alkylesters such as cyclopentyl for glutamic and aspartic acids; benzylethers for serine and threonine; benzyl ethers, substituted benzylethers or 2-bromobenzyloxycarbonyl for tyrosine; p-methylbenzyl,p-methoxybenzyl, acetamidomethyl, benzyl, or t-butylsulfonyl forcysteine; and the indole of tryptophan can either be left unprotected orprotected with a formyl group.

When Fmoc is chosen for the alpha-amine protection usually tert-butylbased protecting groups are acceptable. For instance, Boc can be usedfor lysine, tert-butyl ether for serine, threonine and tyrosine, andtert-butyl ester for glutamic and aspartic acids.

Once the elongation of the peptide, polypeptide or peptidomimetic, orthe elongation and cyclization of a cyclic peptide or peptidomimetic iscompleted all of the protecting groups are removed. For the liquid phasesynthesis the protecting groups are removed in whatever manner asdictated by the choice of protecting groups. These procedures are wellknown to those skilled in the art.

When a solid phase synthesis is used to synthesize a cyclic peptide orpeptidomimetic, the peptide or peptidomimetic should be removed from theresin without simultaneously removing protecting groups from functionalgroups that might interfere with the cyclization process. Thus, if thepeptide or peptidomimetic is to be cyclized in solution, the cleavageconditions need to be chosen such that a free a-carboxylate and a freea-amino group are generated without simultaneously removing otherprotecting groups. Alternatively, the peptide or peptidomimetic may beremoved from the resin by hydrazinolysis, and then coupled by the azidemethod. Another very convenient method involves the synthesis ofpeptides or peptidomimetics on an oxime resin, followed byintramolecular nucleophilic displacement from the resin, which generatesa cyclic peptide or peptidomimetic (Osapay, Profit, and Taylor (1990)Tetrahedron Letters 43, 6121-6124). When the oxime resin is employed,the Boc protection scheme is generally chosen. Then, the preferredmethod for removing side chain protecting groups generally involvestreatment with anhydrous HF containing additives such as dimethylsulfide, anisole, thioanisole, or p-cresol at 0° C. The cleavage of thepeptide or peptidomimetic can also be accomplished by other acidreagents such as trifluoromethanesulfonic acid/trifluoroacetic acidmixtures.

Unusual amino acids used in this invention can be synthesized bystandard methods familiar to those skilled in the art (“The Peptides:Analysis, Synthesis, Biology, Vol. 5, pp. 342-449, Academic Press, NewYork (1981)). N-Alkyl amino acids can be prepared using proceduresdescribed in previously (Cheung et al., (1977) Can. J. Chem. 55, 906;Freidinger et al., (1982) J. Org. Chem. 48, 77 (1982)), which areincorporated herein by reference.

The attachment of linking groups, L_(n), to the MMP inhibitors, Q;chelators or bonding units, C_(h), to the inhibitors, Q, or to thelinking groups, L_(n); and inhibitors bearing a fragment of the linkinggroup to the remainder of the linking group, in combination forming themoiety, (Q)_(d)-L_(n), and then to the moiety C_(h); can all beperformed by standard techniques. These include, but are not limited to,amidation, esterification, alkylation, and the formation of ureas orthioureas. Procedures for performing these attachments can be found inBrinkley, M., Bioconjugate Chemistry 1992, 3(1), which is incorporatedherein by reference.

A number of methods can be used to attach the MMP inhibitors, Q, toparamagnetic metal ion or heavy atom containing solid particles, X², byone of skill in the art of the surface modification of solid particles.In general, the targeting moiety Q or the combination (Q)_(d)L_(n) isattached to a coupling group that react with a constituent of thesurface of the solid particle. The coupling groups can be any of anumber of silanes which react with surface hydroxyl groups on the solidparticle surface, as described in co-pending U.S. patent applicationSer. No. 09/356,178 and can also include polyphosphonates,polycarboxylates, polyphosphates or mixtures thereof which couple withthe surface of the solid particles, as described in U.S. Pat. No.5,520,904.

A number of reaction schemes can be used to attach the MMP inhibitors,Q, to the surfactant microsphere, X³. These are illustrated in followingreaction schemes where S_(f) represents a surfactant moiety that formsthe surfactant microsphere.

Acylation Reaction:S_(f)—C(═O)—Y+Q-NH₂ or ----------->S_(f)—C(═O)—NH-Q Q-OH orS_(f)—C(═O)—O-QY is a leaving group or active esterDisulfide Coupling:S_(f)—SH+Q-SH----------->S_(f)—S—S-QSulfonamide Coupling:S_(f)—S(═O)₂—Y+Q-NH₂----------->S_(f)—S(═O)₂—NH-QReductive Amidation:S_(f)—CHO+Q-NH₂----------->S_(f)—NH-QIn these reaction schemes, the substituents Sf and Q can be reversed aswell.

The linking group L_(n) can serve several roles. First it provides aspacing group between the metal chelator or bonding moiety, C_(h), theparamagnetic metal ion or heavy atom containing solid particle, X², andthe surfactant microsphere, X³, and the one or more of the MMPinhibitors, Q, so as to minimize the possibility that the moietiesC_(h)—X, C_(h)—X¹, X², and X³, will interfere with the interaction ofthe recognition sequences of Q with MMPs associated with cardiovascularpathologies. The necessity of incorporating a linking group in a reagentis dependent on the identity of Q, C_(h)—X, C_(h)—X¹, X², and X³. IfC_(h)—X, C_(h)—X¹, X², and X³, cannot be attached to Q withoutsubstantially diminishing its ability to inhibit MMPs, then a linkinggroup is used. A linking group also provides a means of independentlyattaching multiple inhibitors, Q, to one group that is attached toC_(h)—X, C_(h)—X¹, X², or X³.

The linking group also provides a means of incorporating apharmacokinetic modifier into the pharmaceuticals of the presentinvention. The pharmacokinetic modifier serves to direct thebiodistibution of the injected pharmaceutical other than by theinteraction of the targeting moieties, Q, with the MMPs expressed in thecardiovascular pathologies. A wide variety of functional groups canserve as pharmacokinetic modifiers, including, but not limited to,carbohydrates, polyalkylene glycols, peptides or other polyamino acids,and cyclodextrins. The modifiers can be used to enhance or decreasehydrophilicity and to enhance or decrease the rate of blood clearance.The modifiers can also be used to direct the route of elimination of thepharmaceuticals. Preferred pharmacokinetic modifiers are those thatresult in moderate to fast blood clearance and enhanced renal excretion.

The metal chelator or bonding moiety, Ch, is selected to form stablecomplexes with the metal ion chosen for the particular application.Chelators or bonding moieties for diagnostic radiopharmaceuticals areselected to form stable complexes with the radioisotopes that haveimageable gamma ray or positron emissions, such as ^(99m)Tc, ⁹⁵Tc,¹¹¹In, ⁶²Cu, ⁶⁰Cu, ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y.

Chelators for technetium, copper and gallium isotopes are selected fromdiaminedithiols, monoamine-monoamidedithiols, triamide-monothiols,monoamine-diamide-monothiols, diaminedioximes, and hydrazines. Thechelators are generally tetradentate with donor atoms selected fromnitrogen, oxygen and sulfur. Preferred reagents are comprised ofchelators having amine nitrogen and thiol sulfur donor atoms andhydrazine bonding units. The thiol sulfur atoms and the hydrazines maybear a protecting group which can be displaced either prior to using thereagent to synthesize a radiopharmaceutical or preferably in situ duringthe synthesis of the radiopharmaceutical.

Exemplary thiol protecting groups include those listed in Greene andWuts, “Protective Groups in Organic Synthesis” John Wiley & Sons, NewYork (1991), the disclosure of which is hereby incorporated byreference. Any thiol protecting group known in the art can be used.Examples of thiol protecting groups include, but are not limited to, thefollowing: acetamidomethyl, benzamidomethyl, 1-ethoxyethyl, benzoyl, andtriphenylmethyl.

Exemplary protecting groups for hydrazine bonding units are hydrazoneswhich can be aldehyde or ketone hydrazones having substituents selectedfrom hydrogen, alkyl, aryl and heterocycle. Particularly preferredhydrazones are described in co-pending U.S. Ser. No. 08/476,296 thedisclosure of which is herein incorporated by reference in its entirety.

The hydrazine bonding unit when bound to a metal radionuclide is termeda hydrazido, or diazenido group and serves as the point of attachment ofthe radionuclide to the remainder of the radiopharmaceutical. Adiazenido group can be either terminal (only one atom of the group isbound to the radionuclide) or chelating. In order to have a chelatingdiazenido group at least one other atom of the group must also be boundto the radionuclide. The atoms bound to the metal are termed donoratoms.

Chelators for ¹¹¹In and ⁸⁶Y are selected from cyclic and acyclicpolyaminocarboxylates such as DTPA, DOTA, DO3A, 2-benzyl-DOTA,alpha-(2-phenethyl)1,4,7,10-tetraazazcyclododecane-1-acetic-4,7,10-tris(methylacetic)acid,2-benzyl-cyclohexyldiethylenetriaminepentaacetic acid,2-benzyl-6-methyl-DTPA, and6,6″-bis[N,N,N″,N″-tetra(carboxymethyl)aminomethyl)-4′-(3-amino-4-methoxyphenyl)-2,2′:6′,2″-terpyridine.Procedures for synthesizing these chelators that are not commerciallyavailable can be found in Brechbiel, M. and Gansow, O., J. Chem. Soc.Perkin Trans. 1992, 1, 1175; Brechbiel, M. and Gansow, O., BioconjugateChem. 1991, 2, 187; Deshpande, S., et. al., J. Nucl. Med. 1990, 31, 473;Kruper, J., U.S. Pat. No. 5,064,956, and Toner, J., U.S. Pat. No.4,859,777, the disclosures of which are hereby incorporated by referencein their entirety.

The coordination sphere of metal ion includes all the ligands or groupsbound to the metal. For a transition metal radionuclide to be stable ittypically has a coordination number (number of donor atoms) comprised ofan integer greater than or equal to 4 and less than or equal to 8; thatis there are 4 to 8 atoms bound to the metal and it is said to have acomplete coordination sphere. The requisite coordination number for astable radionuclide complex is determined by the identity of theradionuclide, its oxidation state, and the type of donor atoms. If thechelator or bonding unit does not provide all of the atoms necessary tostabilize the metal radionuclide by completing its coordination sphere,the coordination sphere is completed by donor atoms from other ligands,termed ancillary or co-ligands, which can also be either terminal orchelating.

A large number of ligands can serve as ancillary or co-ligands, thechoice of which is determined by a variety of considerations such as theease of synthesis of the radiopharmaceutical, the chemical and physicalproperties of the ancillary ligand, the rate of formation, the yield,and the number of isomeric forms of the resulting radiopharmaceuticals,the ability to administer said ancillary or co-ligand to a patientwithout adverse physiological consequences to said patient, and thecompatibility of the ligand in a lyophilized kit formulation. The chargeand lipophilicity of the ancillary ligand will effect the charge andlipophilicity of the radiopharmaceuticals. For example, the use of4,5-dihydroxy-1,3-benzene disulfonate results in radiopharmaceuticalswith an additional two anionic groups because the sulfonate groups willbe anionic under physiological conditions. The use of N-alkylsubstituted 3,4-hydroxypyridinones results in radiopharmaceuticals withvarying degrees of lipophilicity depending on the size of the alkylsubstituents.

Preferred technetium radiopharmaceuticals of the present invention arecomprised of a hydrazido or diazenido bonding unit and an ancillaryligand, A_(L1), or a bonding unit and two types of ancillary A_(L1) andA_(L2), or a tetradentate chelator comprised of two nitrogen and twosulfur atoms. Ancillary ligands A_(L1) are comprised of two or more harddonor atoms such as oxygen and amine nitrogen (sp³ hybridized). Thedonor atoms occupy at least two of the sites in the coordination sphereof the radionuclide metal; the ancillary ligand A_(L1) serves as one ofthe three ligands in the ternary ligand system. Examples of ancillaryligands A_(L1) include but are not limited to dioxygen ligands andfunctionalized aminocarboxylates. A large number of such ligands areavailable from commercial sources.

Ancillary dioxygen ligands include ligands that coordinate to the metalion through at least two oxygen donor atoms. Examples include but arenot limited to: glucoheptonate, gluconate, 2-hydroxyisobutyrate,lactate, tartrate, mannitol, glucarate, maltol, Kojic acid,2,2-bis(hydroxymethyl)propionic acid, 4,5-dihydroxy-1,3-benzenedisulfonate, or substituted or unsubstituted 1,2 or 3,4hydroxypyridinones. (The names for the ligands in these examples referto either the protonated or non-protonated forms of the ligands.)

Functionalized aminocarboxylates include ligands that have a combinationof amine nitrogen and oxygen donor atoms. Examples include but are notlimited to: iminodiacetic acid, 2,3-diaminopropionic acid,nitrilotriacetic acid, N,N′-ethylenediamine diacetic acid,N,N,N′-ethylenediamine triacetic acid, hydroxyethylethylenediaminetriacetic acid, and N,N′-ethylenediamine bis-hydroxyphenylglycine. (Thenames for the ligands in these examples refer to either the protonatedor non-protonated forms of the ligands.)

A series of functionalized aminocarboxylates are disclosed by Bridgeret. al. in U.S. Pat. No. 5,350,837, herein incorporated by reference,that result in improved rates of formation of technetium labeledhydrazino modified proteins. We have determined that certain of theseaminocarboxylates result in improved yields of the radiopharmaceuticalsof the present invention. The preferred ancillary ligands A_(L1)functionalized aminocarboxylates that are derivatives of glycine; themost preferred is tricine (tris(hydroxymethyl)methylglycine).

The most preferred technetium radiopharmaceuticals of the presentinvention are comprised of a hydrazido or diazenido bonding unit and twotypes of ancillary designated A_(L1) and A_(L2), or a diaminedithiolchelator. The second type of ancillary ligands A_(L2) are comprised ofone or more soft donor atoms selected from the group: phosphinephosphorus, arsine arsenic, imine nitrogen (sp² hybridized), sulfur (sp²hybridized) and carbon (sp hybridized); atoms which have p-acidcharacter. Ligands A_(L2) can be monodentate, bidentate or tridentate,the denticity is defined by the number of donor atoms in the ligand. Oneof the two donor atoms in a bidentate ligand and one of the three donoratoms in a tridentate ligand must be a soft donor atom. We havedisclosed in co-pending U.S. Ser. No. 08/415,908, and U.S. Ser. No.60/013,360 and 08/646,886, the disclosures of which are hereinincorporated by reference in their entirety, that radiopharmaceuticalscomprised of one or more ancillary or co-ligands A_(L2) are more stablecompared to radiopharmaceuticals that are not comprised of one or moreancillary ligands, A_(L2); that is, they have a minimal number ofisomeric forms, the relative ratios of which do not change significantlywith time, and that remain substantially intact upon dilution.

The ligands A_(L2) that are comprised of phosphine or arsine donor atomsare trisubstituted phosphines, trisubstituted arsines, tetrasubstituteddiphosphines and tetrasubstituted diarsines. The ligands A_(L2) that arecomprised of imine nitrogen are unsaturated or aromaticnitrogen-containing, 5 or 6-membered heterocycles. The ligands that arecomprised of sulfur (sp² hybridized) donor atoms are thiocarbonyls,comprised of the moiety C═S. The ligands comprised of carbon (sphybridized) donor atoms are isonitriles, comprised of the moiety CNR,where R is an organic radical. A large number of such ligands areavailable from commercial sources. Isonitriles can be synthesized asdescribed in European Patent 0107734 and in U.S. Pat. No. 4,988,827,herein incorporated by reference.

Preferred ancillary ligands A_(L2) are trisubstituted phosphines andunsaturated or aromatic 5 or 6 membered heterocycles. The most preferredancillary ligands A_(L2) are trisubstituted phosphines and unsaturated 5membered heterocycles.

The ancillary ligands A_(L2) may be substituted with alkyl, aryl,alkoxy, heterocycle, aralkyl, alkaryl and arylalkaryl groups and may ormay not bear functional groups comprised of heteroatoms such as oxygen,nitrogen, phosphorus or sulfur. Examples of such functional groupsinclude but are not limited to: hydroxyl, carboxyl, carboxamide, nitro,ether, ketone, amino, ammonium, sulfonate, sulfonamide, phosphonate, andphosphonamide. The functional groups may be chosen to alter thelipophilicity and water solubility of the ligands which may affect thebiological properties of the radiopharmaceuticals, such as altering thedistribution into non-target tissues, cells or fluids, and the mechanismand rate of elimination from the body.

Chelators for magnetic resonance imaging contrast agents are selected toform stable complexes with paramagnetic metal ions, such as Gd(III),Dy(III), Fe(III), and Mn(II), are selected from cyclic and acyclicpolyaminocarboxylates such as DTPA, DOTA, DO3A, 2-benzyl-DOTA,alpha-(2-phenethyl)1,4,7,10-tetraazacyclododecane-1-acetic-4,7,10-tris(methylacetic)acid,2-benzyl-cyclohexyldiethylenetriaminepentaacetic acid,2-benzyl-6-methyl-DTPA, and6,6″-bis[N,N,N″,N″-tetra(carboxymethyl)aminomethyl)-4′-(3-amino-4-methoxyphenyl)-2,2′:6′,2″-terpyridine.

There are three key features of the pharmaceuticals of the presentinvention that determine their efficacy: MMP selectivity, inhibitorypotency, typically expressed as the Ki value, and the rate of clearancefrom the blood. Preferred pharmaceuticals of the present invention arecomprised of inhibitors, Q, which exhibit selectivity for MMP-1, MMP-2,MMP-3, MMP-9, or MMP-14 alone or in combination over the other MMPs.Most preferred are comprised of inhibitors, Q, which exhibit selectivityfor MMP-2, MMP-9, or MMP-14 alone or in combination over the other MMPs.Ki values for the preferred pharmaceuticals of the present invention are<100 nM for one or more of MMP-1, MMP-2, MMP-3, MMP-9, or MMP-14. Kivalues for the most preferred pharmaceuticals of the present inventionare <10 nM for one or more of MMP-2, MMP-9, or MMP-14.

The rate of clearance from the blood is of particular importance forcardiac imaging procedures, since the cardiac blood pool is largecompared to the disease foci that one desires to image. For an effectivecardiac imaging agent, the target to background ratios (diseasefoci-to-blood and disease foci-to-muscle) need to be greater or equal to1.5, preferably greater or equal to 2.0, and more preferably evengreater. Preferred pharmaceuticals of the present invention have bloodclearance rates that result in <10% i.d./g at 2 hours post-injection,measured in a mouse model, or <0.5% i.d./g at 2 hours post-injection,measured in a dog model. Most preferred pharmaceuticals of the presentinvention have blood clearance rates that result in <3% i.d./g at 2hours post-injection, measured in a mouse model, or <0.05% i.d./g at 2hours post-injection, measured in a dog model.

The technetium radiopharmaceuticals of the present invention comprisedof a hydrazido or diazenido bonding unit can be easily prepared byadmixing a salt of a radionuclide, a reagent of the present invention,an ancillary ligand A_(L1), an ancillary ligand A_(L2), and a reducingagent, in an aqueous solution at temperatures from 0 to 100° C. Thetechnetium radiopharmaceuticals of the present invention comprised of atetradentate chelator having two nitrogen and two sulfur atoms can beeasily prepared by admixing a salt of a radionuclide, a reagent of thepresent invention, and a reducing agent, in an aqueous solution attemperatures from 0 to 100° C.

When the bonding unit in the reagent of the present invention is presentas a hydrazone group, then it must first be converted to a hydrazine,which may or may not be protonated, prior to complexation with the metalradionuclide. The conversion of the hydrazone group to the hydrazine canoccur either prior to reaction with the radionuclide, in which case theradionuclide and the ancillary or co-ligand or ligands are combined notwith the reagent but with a hydrolyzed form of the reagent bearing thechelator or bonding unit, or in the presence of the radionuclide inwhich case the reagent itself is combined with the radionuclide and theancillary or co-ligand or ligands. In the latter case, the pH of thereaction mixture must be neutral or acidic.

Alternatively, the radiopharmaceuticals of the present inventioncomprised of a hydrazido or diazenido bonding unit can be prepared byfirst admixing a salt of a radionuclide, an ancillary ligand A_(L1), anda reducing agent in an aqueous solution at temperatures from 0 to 100°C. to form an intermediate radionuclide complex with the ancillaryligand A_(L1) then adding a reagent of the present invention and anancillary ligand A_(L2) and reacting further at temperatures from 0 to100° C.

Alternatively, the radiopharmaceuticals of the present inventioncomprised of a hydrazido or diazenido bonding unit can be prepared byfirst admixing a salt of a radionuclide, an ancillary ligand A_(L1), areagent of the present invention, and a reducing agent in an aqueoussolution at temperatures from 0 to 100° C. to form an intermediateradionuclide complex, and then adding an ancillary ligand A_(L2) andreacting further at temperatures from 0 to 100° C.

The technetium radionuclides are preferably in the chemical form ofpertechnetate or perrhenate and a pharmaceutically acceptable oation.The pertechnetate salt form is preferably sodium pertechnetate such asobtained from commercial Tc-99m generators. The amount of pertechnetateused to prepare the radiopharmaceuticals of the present invention canrange from 0.1 mCi to 1 Ci, or more preferably from 1 to 200 mCi.

The amount of the reagent of the present invention used to prepare thetechnetium radiopharmaceuticals of the present invention can range from0.01 μg to 10 mg, or more preferably from 0.5 μg to 200 μg. The amountused will be dictated by the amounts of the other reactants and theidentity of the radiopharmaceuticals of the present invention to beprepared.

The amounts of the ancillary ligands A_(L1) used can range from 0.1 mgto 1 g, or more preferably from 1 mg to 100 mg. The exact amount for aparticular radiopharmaceutical is a function of identity of theradiopharmaceuticals of the present invention to be prepared, theprocedure used and the amounts and identities of the other reactants.Too large an amount of A_(L1) will result in the formation ofby-products comprised of technetium labeled A_(L1) without abiologically active molecule or by-products comprised of technetiumlabeled biologically active molecules with the ancillary ligand A_(L1)but without the ancillary ligand A_(L2). Too small an amount of A_(L1)will result in other by-products such as technetium labeled biologicallyactive molecules with the ancillary ligand A_(L2) but without theancillary ligand A_(L1), or reduced hydrolyzed technetium, or technetiumcolloid.

The amounts of the ancillary ligands A_(L2) used can range from 0.001 mgto 1 g, or more preferably from 0.01 mg to 10 mg. The exact amount for aparticular radiopharmaceutical is a function of the identity of theradiopharmaceuticals of the present invention to be prepared, theprocedure used and the amounts and identities of the other reactants.Too large an amount of A_(L2) will result in the formation ofby-products comprised of technetium labeled A_(L2) without abiologically active molecule or by-products comprised of technetiumlabeled biologically active molecules with the ancillary ligand A_(L2)but without the ancillary ligand A_(L1). If the reagent bears one ormore substituents that are comprised of a soft donor atom, as definedabove, at least a ten-fold molar excess of the ancillary ligand A_(L2)to the reagent of formula 2 is required to prevent the substituent frominterfering with the coordination of the ancillary ligand A_(L2) to themetal radionuclide.

In another embodiment of the current invention, a scintigraphic image ofa radiolabeled MMPI compound would be acquired at the same time as ascintigraphic image of a radiolabeled cardiac perfusion imaging agent.This simultaneous dual isotope imaging would be done by utilizingradioisotopes of the MMPI and perfusion imaging agents which hadspectrally separable gamma emission energies. For example, a Tc99mcardiac perfusion imaging agent (such as Tc99m-Sestamibi) or Tl201 (asThallous Chloride), and an In111-labeled MMPI compound would be imagedsimultaneously with a standard gamma camera. This is possible becausethe Tc99m gamma energy of ˜140 KeV or the Tl201 gamma energy of ˜80 KeVare easily separable from the In111 gamma energies of ˜160 KeV and 250KeV. This simultaneous imaging of cardiac perfusion and extracellularmatrix degradation (as evidenced by MMPI compound localization) isextremely useful for improved anatomic assessment of the location ofMMPI compound distribution in the heart based on the comparison to theperfusion distribution seen on the Tc99m-Sestamibi or Tl201 image. Inaddition, the simultaneous imaging of perfusion and extracellular matrixdegradation allows a more complete assessment of the underlying cardiacdisease, both in terms of blood flow alterations and biochemicalchanges, in a single imaging session on a patient.

The simultaneous dual-isotope imaging of cardiac perfusion andextracellular matrix degradation allows the localization of sites ofvulnerable plaque and cardiac perfusion to be visualized during oneimaging session. In addition, the simultaneous imaging of tissue changesassociated with congestive heart failure (from the MMPI imaging agent)and coronary artery disease (from the perfusion imaging agent) isextremely useful in characterizing the underlying causes of CHF.

The simultaneous imaging of different radioisotopically-labeledradiopharmaceuticals in patients has been reported. For example,Antunes, et al., Am J. Cardiol. 1992; 70: 426-431, have demonstratedthat it is possible to image myocardial infarction with anIn111-antimyosin antibody along with the imaging of cardiac perfusionwith Tl201. However, the dual isotope imaging of the present inventionis new, because it is the first reported approach to the simultaneous,dual isotope imaging of a radiolabeled MMPI compound and a cardiacperfusion imaging compound. The combination of MMPI scintigraphicimaging with perfusion imaging provides the imaging physician with anextraordinary amount of clinical information regarding ischemic coronaryartery disease or congestive heart failure in one imaging session.

Suitable reducing agents for the synthesis of the radiopharmaceuticalsof the present invention include stannous salts, dithionite or bisulfitesalts, borohydride salts, and formamidinesulfinic acid, wherein thesalts are of any pharmaceutically acceptable form. The preferredreducing agent is a stannous salt. The amount of a reducing agent usedcan range from 0.001 mg to 10 mg, or more preferably from 0.005 mg to 1mg.

The specific structure of a radiopharmaceutical of the present inventioncomprised of a hydrazido or diazenido bonding unit will depend on theidentity of the reagent of the present invention used, the identity ofany ancillary ligand A_(L1), the identity of any ancillary ligandA_(L2), and the identity of the radionuclide. Radiopharmaceuticalscomprised of a hydrazido or diazenido bonding unit synthesized usingconcentrations of reagents of <100 μg/mL, will be comprised of onehydrazido or diazenido group. Those synthesized using >1 mg/mLconcentrations will be comprised of two hydrazido or diazenido groupsfrom two reagent molecules. For most applications, only a limited amountof the biologically active molecule can be injected and not result inundesired side-effects, such as chemical toxicity, interference with abiological process or an altered biodistribution of theradiopharmaceutical. Therefore, the radiopharmaceuticals which requirehigher concentrations of the reagents comprised in part of thebiologically active molecule, will have to be diluted or purified aftersynthesis to avoid such side-effects.

The identities and amounts used of the ancillary ligands A_(L1) andA_(L2) will determine the values of the variables y and z. The values ofy and z can independently be an integer from 1 to 2. In combination, thevalues of y and z will result in a technetium coordination sphere thatis made up of at least five and no more than seven donor atoms. Formonodentate ancillary ligands A_(L2), z can be an integer from 1 to 2;for bidentate or tridentate ancillary ligands A_(L2), z is 1. Thepreferred combination for monodentate ligands is y equal to 1 or 2 and zequal to 1. The preferred combination for bidentate or tridentateligands is y equal to 1 and z equal to 1.

The indium, copper, gallium, and yttrium radiopharmaceuticals of thepresent invention can be easily prepared by admixing a salt of aradionuclide and a reagent of the present invention, in an aqueoussolution at temperatures from 0 to 100° C. These radionuclides aretypically obtained as a dilute aqueous solution in a mineral acid, suchas hydrochloric, nitric or sulfuric acid. The radionuclides are combinedwith from one to about one thousand equivalents of the reagents of thepresent invention dissolved in aqueous solution. A buffer is typicallyused to maintain the pH of the reaction mixture between 3 and 10.

The gadolinium, dysprosium, iron and manganese metallopharmaceuticals ofthe present invention can be easily prepared by admixing a salt of theparamagnetic metal ion and a reagent of the present invention, in anaqueous solution at temperatures from 0 to 100° C. These paramagneticmetal ions are typically obtained as a dilute aqueous solution in amineral acid, such as hydrochloric, nitric or sulfuric acid. Theparamagnetic metal ions are combined with from one to about one thousandequivalents of the reagents of the present invention dissolved inaqueous solution. A buffer is typically used to maintain the pH of thereaction mixture between 3 and 10.

The total time of preparation will vary depending on the identity of themetal ion, the identities and amounts of the reactants and the procedureused for the preparation. The preparations may be complete, resultingin >80% yield of the radiopharmaceutical, in 1 minute or may requiremore time. If higher purity metallopharmaceuticals are needed ordesired, the products can be purified by any of a number of techniqueswell known to those skilled in the art such as liquid chromatography,solid phase extraction, solvent extraction, dialysis or ultrafiltration.

Buffers useful in the preparation of metallopharmaceuticals and indiagnostic kits useful for the preparation of said radiopharmaceuticalsinclude but are not limited to phosphate, citrate, sulfosalicylate, andacetate. A more complete list can be found in the United StatesPharmacopeia.

Lyophilization aids useful in the preparation of diagnostic kits usefulfor the preparation of radiopharmaceuticals include but are not limitedto mannitol, lactose, sorbitol, dextran, Ficoll, andpolyvinylpyrrolidine (PVP).

Stabilization aids useful in the preparation of metallopharmaceuticalsand in diagnostic kits useful for the preparation ofradiopharmaceuticals include but are not limited to ascorbic acid,cysteine, monothioglycerol, sodium bisulfite, sodium metabisulfite,gentisic acid, and inositol.

Solubilization aids useful in the preparation of metallopharmaceuticalsand in diagnostic kits useful for the preparation ofradiopharmaceuticals include but are not limited to ethanol, glycerin,polyethylene glycol, propylene glycol, polyoxyethylene sorbitanmonooleate, sorbitan monoloeate, polysorbates,poly(oxyethylene)poly(oxypropylene)poly(oxyethylene) block copolymers(Pluronics) and lecithin. Preferred solubilizing aids are polyethyleneglycol, and Pluronics.

Bacteriostats useful in the preparation of met allopharmaceuticals andin diagnostic kits useful for the preparation of radiopharmaceuticalsinclude but are not limited to benzyl alcohol, benzalkonium chloride,chlorbutanol, and methyl, propyl or butyl paraben.

A component in a diagnostic kit can also serve more than one function. Areducing agent can also serve as a stabilization aid, a buffer can alsoserve as a transfer ligand, a lyophilization aid can also serve as atransfer, ancillary or co-ligand and so forth.

The diagnostic radiopharmaceuticals are administered by intravenousinjection, usually in saline solution, at a dose of 1 to 100 mCi per 70kg body weight, or preferably at a dose of 5 to 50 mCi. Imaging isperformed using known procedures.

The magnetic resonance imaging contrast agents of the present inventionmay be used in a similar manner as other MRI agents as described in U.S.Pat. No. 5,155,215; U.S. Pat. No. 5,087,440; Margerstadt et al., Magn.Reson. Med., 1986, 3, 808; Runge et al., Radiology, 1988, 166, 835; andBousquet et al., Radiology, 1988, 166, 693. Generally, sterile aqueoussolutions of the contrast agents are administered to a patientintravenously in dosages ranging from 0.01 to 1.0 mmoles per kg bodyweight.

For use as X-ray contrast agents, the compositions of the presentinvention should generally have a heavy atom concentration of 1 mM to 5M, preferably 0.1 M to 2 M. Dosages, administered by intravenousinjection, will typically range from 0.5 mmol/kg to 1.5 mmol/kg,preferably 0.8 mmol/kg to 1.2 mmol/kg. Imaging is performed using knowntechniques, preferably X-ray computed tomography.

The ultrasound contrast agents of the present invention are administeredby intravenous injection in an amount of 10 to 30 μL of the echogenicgas per kg body weight or by infusion at a rate of approximately 3μL/kg/min. Imaging is performed using known techniques of sonography.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not intended to be limitingthereof.

EXAMPLE 1 Synthesis of2-{[5-(3-{2-[(6-Hydroxycarbamoyl-7-isobutyl-8-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10-carbonyl)-amino]-acetylamino}-propylcarbamoyl)-pyridin-2-yl]-hydrazonomethyl}-benzenesulfonicacid

A. Preparation of[3-(2-Benzyloxycarbonylamino-acetylamino)-propyl]-carbamic acidtert-butyl ester

To 3 grams of (3-Amino-propyl)-carbamic acid tert-butyl ester in 15 mlof dimethylformamide was added 3 grams of N-benzyloxycarbonyl glycine,4.7 mL of N-methylmorpholine and 5.06 grams of TBTU. The reaction wascooled to 0 degrees C. for 30 minutes then allowed to stir at roomtemperature overnight. The volatiles were removed under reduced pressureand the resulting material was dissolved in ethyl acetate and washedwith 10% citric acid. The aqueous was extracted an additional two timeswith ethyl acetate, combined and washed with water, saturated aqueoussodium bicarbonate, water, brine and dried over MgSO₄. The volatileswere removed under reduced pressure and the resulting material wascrystallized from EtOAc/hexane affording 4.55 grams of the desiredproduct as a tan solid. LRMS found 388.3=(M+Na)⁺

B. Preparation of [3-(2-Amino-acetylamino)-propyl]-carbamic acidtert-butyl ester

To 4.16 grams of the compound from Example 1A in 25 mL of methanol wasadded 0.5 grams of 10% Pd—C. The reaction was stirred under H₂ (balloon)for 2 hours. The reaction was filtered through a 0.45 μM PTFE filter andthe volatiles were removed under reduced pressure affording 2.5 grams ofthe desired product. LRMS found 232.3 (M+H)⁺¹.

C. Preparation of3-{2-[(6-Benzyloxycarbamoyl-7-isobutyl-8-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10-carbonyl)-amino}-propyl)-carbamicacid tert-butyl ester

To 0.25 grams of6-Benzyloxycarbamoyl-7-isobutyl-9-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10-carboxylicacid in 10 mL of dimethylformamide was added 0.17 ml N-methylmorpholineand 0.217 grams of TBTU. After 10 minutes 0.359 grams of the compoundfrom Example 1B was added. The reaction was allowed to stir at roomtemperature overnight, then it was heated at 70 degrees C. for 30minutes. The volatiles were removed under reduced pressure and theresulting material was dissolved in EtOAc, washed with 10% aqueouscitric acid, water, saturated NaHCO₃, brine and dried over MgSO₄. Theresulting material was chromatographed on silica gel eluting with 2%MeOH/CHCl₃ affording 0.274 grams of the desired product.

D. Preparation of3-{2-[(6-Hydroxycarbamoyl-7-isobutyl-8-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10-carbonyl)-amino}-propyl)-carbamicacid tert-butyl ester

To 0.035 grams of the compound from Example 1C in 5 mL of methanol wasadded 0.050 grams of 5% Pd/BaSO₄. The reaction was stirred underhydrogen (balloon) for 2 hours, then filtered through a 0.45 μM PTFEfilter and the volatiles were removed under reduced pressure affording0.031 grams of the desired compound. LRMS found 604.4 (M−H)⁻¹.

E. Preparation of7-Isobutyl-8-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-6,10-dicarboxylicacid 10-{[(3-amino-propylcarbamoyl)-methyl]-amide} 6-hydroxyamidetrifluoroacetic acid salt

To 0.025 grams of the compound form Example 1D was added 1 mL oftrifluoroacetic acid. The reaction was stirred 1 hour and the volatileswere removed under reduced pressure affording 0.017 grams of the desiredcompound. LRMS found 506.4 (M+H)⁺¹.

F. Preparation of2-{[5-(3-{2-[(6-Hydroxycarbamoyl-7-isobutyl-8-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10-carbonyl)-amino]-acetylamino}-propylcarbamoyl)-pyridin-2-yl]-hydrazonomethyl}-benzenesulfonicacid

To a stirred solution of 0.050 grams of the compound from Example 1E wasadded 0.031 mL of N-methylmorpholine and 0.035 grams of6-[N″-(2-Sodio-sulfo-benzylidene)-hydrazino]-nicotinic acid2,5-dioxo-pyrrolidin-1-yl ester. The reaction was stirred at ambienttemperature overnight. Volatiles were removed under reduced pressure andthe resulting material was purified by reverse phase HPLC affording 0.08grams of the desired compound. LRMS found 807 (M−H)⁻¹.

EXAMPLE 2 Synthesis of2-{[5-(4-{[(6-Hydroxycarbamoyl-7-isobutyl-8-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10-carbonyl)-amino]-methyl}-benzylcarbamoyl)-pyridin-2-yl]-hydrazonomethyl}-benzenesulfonicacid

A. Preparation of (4-Aminomethyl-benzyl)carbamic acid tert-butyl ester

To a stirred solution of 5.3 grams of p-xylenediamine 10 in 20 mL ofdimethylformamide was added a solution of 2.12 grams ofdi-tert-butyl-dicarbonate in 50 mL of dimethylformamide by syringe pumpover 1 hour. After stirring an additional 10 minutes the volatiles wereremoved under reduced pressure and the resulting material waschromatographed on silica gel eluting with 5% MeOH/CHCl₃ affording 2grams of the desired compound. LRMS found 237.2 (M+H)⁺¹.

B. Preparation of(4-{[(6-Benzyloxycarbamoyl-7-isobutyl-8-oso-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10-carbonyl)-amino]-methyl}-benzyl)-carbamicacid tert-butyl ester

To 0.20 grams of6-Benzyloxycarbamoyl-7-isobutyl-9-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10-carboxylicacid in 5 mL of dimethylformamide was added 0.18 mL ofn-methylmorpholine and 0.173 grams of TBTU. After stirring 20 minutes0.293 grams of the compound from Example 2A was added. After stirring atambient temperature overnight the reaction was heated to 80 degrees C.for 30 minutes. The volatiles were removed under reduced pressure andthe resulting material was dissolved in EtOAc, washed with 10% aqueouscitric acid, water, saturated NaHCO₃, brine and dried over MgSO₄. Thevolatiles were removed under reduced pressure affording 0.296 grams ofthe desired compound. LRMS found 699.4 (M−H)⁻¹.

C. Preparation of(4-{[(6-Hydroxycarbamoyl-7-isobutyl-8-oso-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10-carbonyl)-amino]-methyl}-benzyl)-carbamicacid tert-butyl ester

To 0.275 grams of the compound from Example 2B in 20 mL of methanol wasadded 0.50 grams of pre-hydrogenated 5% pd-BaSO₄. The reaction wasstirred 3 hours under H₂ (Balloon) at which time an additional portionof 0.25 grams of 5% Pd—BaSO₄ was added and the stirring was continuedfor another hour. The mixture was filtered through a 0.45 uM PTFE filterand the volatiles were removed under reduced pressure affording 0.24grams of the desired compound. LRMS found 609.4 (M−H)⁻¹.

D. Preparation of7-Isobutyl-8-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-6,10-dicarboxylicacid 10-(4-aminomethyl-benzylamice) 6-hydroxyamide trifluoroacetic acidsalt

To 0.225 grams of the compound from Example 2C in 5 mL of CH₂Cl₂ wasadded 2 mL of trifluoroacetic acid. The reaction was stirred one hour atambient temperature. The volatiles were removed under reduced pressureaffording the desired compound. LRMS found 509.4 (M−H)⁻¹.

E. Preparation of2-{[5-(4-{[(6-Hydroxycarbamoyl-7-isobutyl-8-oxo-2-oxa-9-aza-bicyclo[10.2.2]hexadeca-1(15),12(16),13-triene-10-carbonyl)-amino]-methyl}-benzylcarbamoyl)-pyridin-2-yl]-hydrazonomethyl}-benzenesulfonicacid

To 0.050 grams of the compound from Example 2D in 1 mL ofdimethylformamide was added 0.031 mL of N-methylmorpholine and 0.035grams of of 6-[N″-(2-Sodio-sulfo-benzylidene)-hydrazino]-nicotinic acid2,5-dioxo-pyrrolidin-1-yl ester. After stirring overnight an ambienttemperature the volatiles were removed under reduced pressure and theresulting material was purified by reverse phase HPLC affording 0.06grams the desired compound. LRMS found 814 (M+H)⁺¹.

EXAMPLE 3 Synthesis of2-[7-({N-[3-(2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}acetylamino)propyl]carbamoyl}methyl)-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl]aceticacid

A solution of the commercially (Macrocyclics) available DOTA tri-t-butylester (1.5 mmol) and Hunig's base (6 mmol) in anhydrous DMF are treatedwith HBTU (1.25 mmol) and allowed to react for 15 min at ambienttemperatures under nitrogen.2-{[7-(N-Hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}-N-(3-aminopropyl)acetamideTFA salt (1 mmol) is added to this solution and stirring is continued atambient temperatures under nitrogen for 18 h. The DMF is removed undervacuum and the resulting residue is triturated in ethyl acetate ordiethyl ether and filtered. If necessary, the crude is purified bypreparative HPLC on a C18 column using a water:ACN:0.1% TFA gradient andthe product fraction is lyophilized to give the DOTA-conjugate. The DOTAconjugate is stirred in degassed TFA at room temperature under nitrogenfor 2 h. The solution is concentrated and the resulting residue ispurified by preparative HPLC on a C18 column using a water:ACN:0.1% TFAgradient. The product fraction is lyophilized to give the titlecompound.

EXAMPLE 4 Synthesis of2-{7-[(N-{[4-({[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-carbonylamino}methyl)phenyl]methyl}carbamoyl)methyl]-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl}aceticacid

A solution of the commercially (Macrocyclics) available DOTA tri-t-butylester (1.5 mmol) and Hunig's base (6 mmol) in anhydrous DMF are treatedwith HBTU (1.25 mmol) and allowed to react for 15 min at ambienttemperatures under nitrogen. [7-(N-Hydroxycarbamoyl)(3S,6R,7S)-4-aza-6(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-N-{[4-(aminomethyl)phenyl]methyl}carboxamideTFA salt (1 mmol) is added to this solution and stirring is continued atambient temperatures under nitrogen for 18 h. The DMF is removed undervacuum and the resulting residue is triturated in ethyl acetate ordiethyl ether and filtered. If necessary, the crude is purified bypreparative HPLC on a C18 column using a water:ACN:0.1% TFA gradient andthe product fraction is lyophilized to give the DOTA-conjugate.

The DOTA conjugate is stirred in degassed TFA at room temperature undernitrogen for 2 h. The solution is concentrated and the resulting residueis purified by preparative HPLC on a C18 column using a water:ACN:0.1%TFA gradient. The product fraction is lyophilized to give the titlecompound.

EXAMPLE 5 Synthesis of2-(7-{[N-(1-{N-[3-(2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}acetylamino)propyl]carbamoyl}-2-sulfoethyl)carbamoyl]methyl}-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl)aceticacid

A. Preparation ofN-[3-(2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}acetylamino)propyl]-2-aminopropanesulfonicacid

2-{[7-(N-Hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3yl]carbonylamino}-N-(3-aminopropyl)acetamideTFA salt (1 mmol) is dissolved in anhydrous DMF, and treated with theN-hydroxysuccinimide ester (1.5 mmol) of Boc-cysteic acid (as describedin Liebigs Ann. Chem. 1979, 776-783) and Hunig's base. The solution isstirred at ambient temperatures under nitrogen for 18 h, and the DMF isremoved under vacuum. The resulting residue is purified by preparativeHPLC on a C18 column using a water:ACN:0.1% TFA gradient. The productfraction is lyophilized to give a solid which is dissolved in degassedTFA and stirred at ambient temperatures for 30 min. The solution isconcentrated under vacuum, and the resulting residue is dissolved in 50%ACN and lyophilized to give the boc deprotected productN-[3-(2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}acetylamino)propyl]-2-aminopropanesulfonicacid.

B. Preparation of2-(7-{[N-(1-{N-[3-(2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}acetylamino)propyl]carbamoyl}-2-sulfoethyl)carbamoyl]methyl}-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl)aceticacid.

A solution of the commercially (Macrocyclics) available DOTA tri-t-butylester (1.5 mmol) and Hunig's base (6 mmol) in anhydrous DMF are treatedwith HBTU (1.25 mmol) and allowed to react for 15 min at ambienttemperatures under nitrogen.N-[3-(2-{[7-(N-Hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}acetylamino)propyl]-2-aminopropanesulfonicacid is added to this solution and stirring is continued at ambienttemperatures under nitrogen for 18 h. The DMF is removed under vacuumand the resulting residue is triturated in ethyl acetate or diethylether and filtered. If necessary, the crude is purified by preparativeHPLC on a C18 column using a water:ACN:0.1% TFA gradient and the productfraction is lyophilized to give the DOTA-conjugate.

The DOTA conjugate is stirred in degassed TFA at room temperature undernitrogen for 2 h. The solution is concentrated and the resulting residueis purified by preparative HPLC on a C18 column using a water:ACN:0.1%TFA gradient. The product fraction is lyophilized to give the titlecompound.

EXAMPLE 6 Synthesis of2-[7-({N-[1-(N-{[4-({[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-carbonylamino}methyl)phenyl]methyl}carbamoyl)-2-sulfoethyl]carbamoyl}methyl)-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl]aceticacid

A. Preparation ofN-{[4-({[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-carbonylamino}methyl)phenyl]methyl}-2-aminopropanesulfonicacid

[7-(N-Hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-N-{[4-(aminomethyl)phenyl]methyl}carboxamideTFA salt (1 mmol) is dissolved in anhydrous DMF, and treated with theN-hydroxysuccinimide ester (1.5 mmol) of Boc-cysteic acid (as describedin Liebigs Ann. Chem. 1979, 776-783) and Hunig's base. The solution isstirred at ambient temperatures under nitrogen for 18 h, and the DMF isremoved under vacuum. The resulting residue is purified by preparativeHPLC on a C18 column using a water:ACN:0.1% TFA gradient. The productfraction is lyophilized to give a solid, which is dissolved in degassedTFA and stirred at ambient temperatures for 30 min. The solution isconcentrated under vacuum, and the resulting residue is dissolved in 50%ACN and lyophilized to give the boc deprotected productN-{[4-({[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-carbonylamino}methyl)phenyl]methyl}-2-aminopropanesulfonicacid.

B. Preparation of2-[7-({N-[1-(N-{[4-({[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-carbonylamino}methyl)phenyl]methyl}carbamoyl)-2-sulfoethyl]carbamoyl}methyl)-1,4,7,10-tetraaza-4,10-bis(carboxymethyl)cyclododecyl]aceticacid.

A solution of the commercially (Macrocyclics) available DOTA tri-t-butylester (1.5 mmol) and Hunig's base (6 mmol) in anhydrous DMF are treatedwith HBTU (1.25 mmol) and allowed to react for 15 min at ambienttemperatures under nitrogen.N-{[4-({[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-carbonylamino}methyl)phenyl]methyl}-2-aminopropanesulfonicacid is added to this solution and stirring is continued at ambienttemperatures under nitrogen for 18 h. The DMF is removed under vacuumand the resulting residue is triturated in ethyl acetate or diethylether and filtered. If necessary, the crude is purified by preparativeHPLC on a C18 column using a water:ACN:0.1% TFA gradient and the productfraction is lyophilized to give the DOTA-conjugate.

The DOTA conjugate is stirred in degassed TFA at room temperature undernitrogen for 2 h. The solution is concentrated and the resulting residueis purified by preparative HPLC on a C18 column using a water:ACN:0.1%TFA gradient. The product fraction is lyophilized to give the titlecompound.

EXAMPLE 7 Synthesis of2-({2-[({N-[3-(2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}acetylamino)propyl]carbamoyl}methyl)(carboxymethyl)amino}ethyl){2-[bis(carboxymethyl)amino]ethyl}amino]aceticacid

To a solution of2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}-N-(3-aminopropyl)acetamideTFA salt (1 mmol) in DMF (20 mL) is added triethylamine (3 mmol). Thissolution is added dropwise over 4 h to a solution ofdiethylenetriaminepentaacetic dianhydride (3 mmol) in DMF (20 mL) andmethyl sulfoxide (20 mL). The reaction mixture is then stirred for 16 h,concentrated to an oil under high vacuum and purified by preparativeHPLC to give the title compound.

EXAMPLE 8 Synthesis of2-[(2-{[(N-{[4-({[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-carbonylamino}methyl)phenyl]methyl}carbamoyl)methyl](carboxymethyl)amino}ethyl){2-[bis(carboxymethyl)amino]ethyl}amino]aceticacid

To a solution of[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-N-{[4-(aminomethyl)phenyl]methyl}carboxamideTFA salt (1 mmol) in DMF (20 mL) is added triethylamine (3 mmol). Thissolution is added dropwise over 4 h to a solution ofdiethylenetriaminepentaacetic dianhydride (3 mmol) in DMF (20 mL) andmethyl sulfoxide (20 mL). The reaction mixture is then stirred for 16 h,concentrated to an oil under high vacuum and purified by preparativeHPLC to give the title compound.

EXAMPLE 9 Synthesis ofN-[3-(2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}acetylamino)propyl]-4,5-bis[2-(ethoxyethylthio)acetylamino]pentanamide

To a solution of2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}-N-(3-aminopropyl)acetamideTFA salt (1 mmol) and triethylamine (3 mmol) in DMF is added2,3,5,6-tetrafluorophenyl4,5-bis(S-1-ethoxyethyl-mercapto-acetamido)pentanoate (1.1 mmol), andthe reaction mixture is stirred for 18 hours. DMF is removed in vacuoand the crude residue is triturated with ethyl acetate. The product isfiltered, dried, and if necessary, further purified by preparative HPLCto give the title compound.

EXAMPLE 10 Synthesis ofN-{[4-({[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}methyl)-phenyl]methyl}-4,5-bis[2-(ethoxyethylthio)acetylamino]-pentanamide

To a solution of[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-N-{[4-(aminomethyl)phenyl]-methyl}carboxamideTFA salt (1 mmol) and triethylamine (3 mmol) in DMF is added2,3,5,6-tetrafluorophenyl4,5-bis(S-1-ethoxyethyl-mercapto-acetamido)pentanoate (1.1 mmol), andthe reaction mixture is stirred for 18 hours. DMF is removed in vacuoand the crude residue is triturated with ethyl acetate. The product isfiltered, dried, and if necessary, further purified by preparative HPLCto give the title compound.

EXAMPLE 11 Synthesis of1-(1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamino)-α,ω-dicarbonylPEG₃₄₀₀-2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}-N-(3-aminopropyl)acetamideconjugate

To solution of the commercially available (Shearwater Polymers)succinimidyl ester, DSPE-PEG-NHS ester (1 mmol) in 25 ml chloroform isadded 2-{[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]carbonylamino}-N-(3-aminopropyl)acetamideTFA salt (1 mmol). Sodium carbonate (1 mmol) and sodium sulfate (1 mmol)are added and the solution is stirred at room temperature under nitrogenfor 18 h. The solvent is removed in vacuo and the crude product ispurified using preparative HPLC to obtain the title compound.

EXAMPLE 12 Synthesis of1-(1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamino)-α,ω-dicarbonylPEG₃₄₀₀-[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-N-{[4-(aminomethyl)phenyl]methyl}carboxamideconjugate

To solution of the commercially available (Shearwater Polymers)succinimidyl ester, DSPE-PEG-NHS ester (1 mmol) in 25 ml chloroform isadded[7-(N-hydroxycarbamoyl)(3S,6R,7S)-4-aza-6-(2-methylpropyl)-11-oxa-5-oxobicyclo[10.2.2]hexadeca-1(15),12(16),13-trien-3-yl]-N-{[4-(aminomethyl)phenyl]methyl}carboxamideTFA salt (1 mmol). Sodium carbonate (1 mmol) and sodium sulfate (1 mmol)are added and the solution is stirred at room temperature under nitrogenfor 18 h. The solvent is removed in vacuo and the crude product ispurified using preparative HPLC to obtain the title compound.

EXAMPLES 13 AND 14 Synthesis of ^(99m)Tc Complexes

To a lyophilized vial containing 4.84 mg TPPTS, 6.3 mg tricine, 40 mgmannitol, succinic acid buffer, pH 4.8, and 0.1% Pluronic F-64surfactant, was added 0.75-1.1 mL sterile water for injection, 0.2-0.45mL (20-40 μg) of the compounds of Examples 1 and 2, respectively, indeionized water or 50% aqueous ethanol, and 0.2-0.4 mL of ^(99m)TcO₄ ⁻(50-120 mCi) in saline. The reconstituted kit was heated in a 100° C.water bath for 10-15 minutes, and was allowed to cool 10 minutes at roomtemperature. A sample was then analyzed by HPLC.

HPLC Method for Example 13

-   Column: Zorbax C18, 25 cm×4.6 mm-   Flow rate: 1.0 mL/min-   Solvent A: 10 mM sodium phosphate buffer, pH 6.0

Solvent B: 100% CH₃CN t (min) 0 20 21 30 31 40 % Solvent B 0 25 75 75 00HPLC Method for Example 14

-   Column: Zorbax C18, 25 cm×4.6 mm-   Flow rate: 1.0 mL/min-   Solvent A: 10 mM sodium phosphate buffer, pH 6.0

Solvent B: 100% CH₃CN t (min) 0 20 21 26 27 40 % Solvent B 0 25 75 75 00

Example # Reagent Ex. # Ret. Time (min) % Yield 13 1 7.8 79 14 2 16.7 81

EXAMPLE 15 Synthesis of2-[2-({5-[N-(5-(N-hydroxycarbamoyl)(5R)-5-{3-[4-(3,4-dimethoxyphenoxy)phenyl]-3-methyl-2-oxopyrrolidinyl}pentyl)carbamoyl](2-pyridyl)}amino)(1Z)-2-azavinyl]benzenesulfonicacid

The title compound can be synthesized as shown in Scheme I from thestarting materials described in the following patent applications whichare hereby incorporated by reference into this patent application: U.S.patent application Ser. Nos. 09/165,747, 08/743,439, 09/134,484,09/247,675, 09/335,086, 09/312,066, 09/311,168, 60/127,594, and60/127,635.

EXAMPLE 16 Synthesis of2-(2-{[5-(N-{3-[3-(N-hydroxycarbamoyl)(4S)-4-({4-[(4-methylphenyl)methoxy]piperidyl}carbonyl)piperidyl]-3-oxopropyl}-carbamoyl)(2-pyridyl)]amino}(1Z)-2-azavinyl)benzenesulfonicacid

The title compound can be synthesized as shown in Scheme II from thestarting materials described in the following patent applications whichare hereby incorporated by reference into this patent application: U.S.patent application Ser. Nos. 09/165,747, 08/743,439, 09/134,484,09/247,675, 09/335,086, 09/312,066, 09/311,168, 60/127,594, and60/127,635.

EXAMPLE 17 Synthesis of N-BOC-Glycine-(3-carbobenzyloxyamido)propylamide

Di-isopropylethylamine (7.0 mL, 40 mmol) was added to a suspension ofN-t-butyloxycarbonylglycine N-hydroxysuccinimide ester (5.56 g, 20 mmol)and N-carbobenzyloxy-1,3-diaminopropane hydrochloride (5.0 g, 20 mmol)in dichloromethane (50 ml). The solution became clear over severalminutes. After 30 minutes, additional of N-t-butyloxycarbonylglycineN-hydroxysuccinimide ester (0.275 g, 1 mmol) was added. The solution wasextracted with water, followed by saturated aqueous NaHCO₃, then by 0.5N HCl. The dichloromethane solution was filtered through a short columnof Na₂SO₄ and evaporated in vacuo to obtain 4.1 g (56%) ofN-BOC-Glycine-(3-carbobenzyloxyamido)propylamide. MS: m/e=366 (M+H⁺),310 (M-C₄H₉+H⁺), 266 (M-BOC+H⁺).

Synthesis of Glycine-(3-carbobenzyloxyamido)propylamide

N-BOC-Glycine-(3-carbobenzyloxyamido)propylamide (260 mg, 0.71 mmol) wasdissolved in dichloromethane (3 mL) and trifluoroacetic acid (0.5 mL)added. After 20 minutes, the solution was evaporated in vacuo, dissolvedin dichloromethane (2 mL) and evaporated in vacuo to obtain the crudeproduct which was used directly in the next reaction. MS: m/e=266(M+H⁺).Synthesis of 17a:

Di-isopropylethylamine (0.25 mL, 1.4 mmol) was added to a mixture of 1(300 mg, 0.69 mmol) and HBTU (270 mg, 0.71 mmol) in dichloromethane (5mL). Dimethylformamide (1 mL) was added to obtain a clear solution.After 30 minutes, a solution ofGlycine-(3-carbobenzyloxyamido)propylamide (−0.71 mmol) anddi-isopropylethyl amine (0.25 mL, 1.4 mmol) in dichloromethane (1 mL)was added. The reaction mixture was extracted twice with 0.5 N HCl, onceeach with saturated aqueous NaCl, 1.0 N NaOH, saturated aqueous NaCl,and saturated aqueous NaHCO₃. The dichloromethane solution was filteredthrough a short column of Na₂SO₄ and evaporated in vacuo to obtain crude17b (599 mg, 127%). MS: m/e=681 (M+H⁺). 1:

Synthesis of 17b:

Trifluoroacetic acid (1 mL) was added to a solution of 17a (599 mg) indichloromethane (5 mL) and allowed to stand at room temperatureovernight. The solution was evaporated in vacuo, dissolved indichloromethane (2 mL) and evaporated in vacuo to obtain 3 (714 mg). MS:m/e=625 (M+H⁺).Synthesis of 17c:

A mixture of 3 (˜0.3 g, ˜0.5 mmol) and 10% Pd/C (25 mg) in ethanol (5mL) was stirred under hydrogen (1 atm) for 2.5 hours. Disappearance of17b was accompanied by the appearance of two peaks in the HPLC-MSchromatogram, both of which exhibited base peaks at m/e=491 amu,consistent with (M+H⁺) for the loss of carbobenzyloxy group from 26b.The reaction mixture was filtered through Celite and evaporated in vacuoto obtain 17c.Synthesis of 17d:

A mixture ofN,N-bis[2-bis(1,1′-dimethylethoxy)-2-oxoethyl]-amino]ethyl]glycine (487mg, 0.788 mmol), HBTU (288 mg, 0.760 mmol) and di-isopropylethyl amine(0.4 mL, 2.3 mmol) in dimethylformamide (4 mL) was stirred at roomtemperature. A solution of 4 (˜0.5 mmol) in dimethylformamide (2 mL) wasadded in one portion. After 2 hours, ˜⅔ of the solution was removed,partitioned between dichloromethane and 0.5 M HCl. The organic phaseextracted once with 0.5 M HCl, then with saturated aqueous NaCl,filtered through a column of Na₂SO₄, and evaporated in vacuo to obtaincrude 17d. MS: m/e 1090 (M+H⁺), 546 (M+2H)⁺²Synthesis of 17e

The remaining ⅓ of the reaction mixture 17d was treated with HBTU (75mg, 0.20 mmol) and allowed to stir for 20 minutes. A solution preparedfrom hydroxylamine hydrochloride (50 mg, 0.70 mmol) anddi-isopropylethylamine (0.15 mL, 0.86 mmol) in dimethylformamide (0.5mL) was added in one portion. The reaction mixture was partitionedbetween dichloromethane and 0.5 M HCl. The organic phase was extractedwith saturated aqueous NaCl, then with saturated aqueous NaHCO₃. TheNaHCO₃ phase was back-extracted with dichloromethane, the combinedorganic extracts filtered through a column of Na₂SO₄, and evaporated invacuo. The crude product was purified by reverse-phase HPLC to obtain 14mg of 27e. MS: m/e 1105 (M+H⁺), 553 (M+2H)⁺²Synthesis of 17f:

A solution of 17e in trifluoroacetic acid (0.5 mL) and dichloromethane(2 mL) was allowed to stand at room temperature overnight. The solutionwas evaporated in vacuo, dissolved in acetonitrile-water and purified byreverse-phase HPLC to obtain 3.4 mg of 17f. MS: m/e 881 (M+H⁺), 441(M+2H)⁺²

Utility

The pharmaceuticals of the present invention are useful for imaging ofcardiovascular disease processes involving the degradation of theextracellular matrix. The radiopharmaceuticals of the present inventioncomprised of a gamma emitting isotope are useful for imaging ofcardiovascular pathological processes involving the degradation of theextracellular matrix, including atherosclerosis, congestive heartfailure, and restenosis of blood vessels after angioplasty.

The compounds of the present invention comprised of one or moreparamagnetic metal ions selected from gadolinium, dysprosium, iron, andmanganese, are useful as contrast agents for magnetic resonance imaging(MRI) of cardiovascular pathological processes involving extracellularmatrix degradation.

The compounds of the present invention comprised of one or more heavyatoms with atomic number of 20 or greater are useful as X-ray contrastagents for X-ray imaging of cardiovascular pathological processesinvolving extracellular matrix degradation.

The compounds of the present invention comprised of an echogenic gascontaining surfactant microsphere are useful as ultrasound contrastagents for sonography of cardiovascular pathological processes involvingextracellular matrix degradation.

Representative compounds of the present invention were tested in the oneor more of the following in vitro assays and were found to be active.

Matrix Metalloproteinase Assays for MMP-1 (collagenase-1), MMP-2(gelatinase A), MMP-3 (stromelysin-1), MMP-8 (collagenase-2), MMP-9(gelatinase B), MMP-13 (collagenase-3), MMP-14 (membrane type 1 MMP),MMP-15 (membrane type 2 MMP), and MMP-16 (membrane type 3 MMP).

A. Reagents

-   1. MCA peptide substrate: Mca-Pro-Leu-Gly-Leu-Dpa-Ala-NH2. Peptide    stocks are stored at −70 C in DMSO at 20 mM. Dilute peptide in 1×    reaction buffer to a working concentration of 14 uM on day of use.-   2. Enzyme buffer. 50 mM Tricine, 0.05% Brij-35, 400 mM NaCl, 10 mM    CaCl2, 0.02% NaN3, pH 7.5.-   3. Reaction buffer. 50 mM Tricine, 10 mM CaCl₂, 0.02% NaN3, pH 7.5-   4. Compounds. Stock compounds are at 10 mM in DMSO. Dilutions are    done in buffer.-   5. Plates. microfluor W flat bottom plates (Dynex Inc. Cat. #7905)    B. Assay-   1. To 96 well fluorescent assay plates add 2 uL of DMSO control or    compound dilutions to wells.-   2. Add 20 uL of EDTA (0.5M) to each quench well.-   3. Add 50 uL of enzyme at the appropriate concentration.-   4. Add 150 uL of the MCA peptide at final concentration of 10 uM.-   5. Incubate each plate for 1 hour at room temperature on an orbital    shaker.-   6. Add 20 uL of EDTA (0.5 M) to each test well to quench the    reaction.-   7. Read each plate at 330 nm excitation, 440 nm emission (Dynx plate    reader).-   8. Subtract each quench value from the corresponding test value.-   9. % inhibition=100−(sample fluorescence/control fluorescence)×100.    TACE Assay    A. Reagents-   1. MCA Peptide substrate: Mca-PLAQAV(Dpa)RSSSR-NH2. Peptide stocks    are stored at −70 C in DMSO at 20 mM. Dilute peptide stock in    reaction buffer to a working concentration of 20 uM on day of use.-   2. Reaction buffer. 50 mM Tricine, 100 mM NaCl, 10 mM CaCl2, 1 mM    ZnCl2, pH 7.5.-   3. Compounds. Stock compounds are at 10 mM in DMSO-   4. Plates. black Packard Optiplate (Cat. # HTRF-96)-   5. Cytofluor Multi-well Plate Reader (Series 4000)    B. Assay-   1. Initiate assay by adding 2 nM TACE to buffered solutions    containing 10 □M MCA peptide substrate in the presence of increasing    concentrations of compound.-   2. Add 20 uL of EDTA (0.5M) to each quench well.-   3. Total volume is 300 uL in each well.-   4. Incubate the reaction mixtures for 1 hour at 28 C on an orbital    shaker.-   5. Add 20 uL of EDTA (0.5M) to each test well to quench the    reaction.-   6. Read each plate at 330 nM excitation, 395 nm emission-   7. Subtract each quench value from the corresponding test value.-   8. % inhibition=100−(sample fluorescence/control fluorescence)×100    In Vivo Models

Cardiovascular disease models that can be used to assess the diagnosticradiopharmaceuticals, magnetic resonance, X-ray and ultrasound contrastagents of the present invention are reviewed in J. Nucl. Cardiol., 1998,5, 167-83. There are several well established rabbit models ofatherosclerosis; one model produces predominantly proliferating smoothmuscle cells by balloon deendothelialization of infradiaphragmaticabdominal aorta to simulate restenotic lesions; another model thatproduces simulated advanced human atherosclerotic plaque by balloondeendothelialization followed by a high cholesterol diet.

A model of congestive heart failure is described in Am. J. Physiol.,1998, 274, H1516-23. In general, Yorkshire pigs are randomly assigned toundergo 3 wks of rapid atrial pacing at 240 beats/min. or to be shamcontrols. The pigs are chronically instrumented to measure leftventricular function in the conscious state. The pigs are anesthetized.

A shielded stimulating electrode is sutured onto the left atrium,connected to a modified programmable pace maker and buried in asubcutaneous pocket. The pericardium is closed loosely, the thoracotomyis closed, and the pleural space is evacuated of air. After a recoveryperiod of 7-10 days, the pacemaker is activated in the animals selectedto undergo chronic rapid pacing. The animals are sedated, the pacemakeris deactivated (pacing groups only. After a 30 min stabilization period,indexes of LV function and geometry are determined (by echocardiographyas a control) by injecting the radiolabeled compound. Forbiodistribution, the animals are anesthetized, the heart extirpate andthe LV apex and midventricular regions are evaluated.

A rat model of reversible coronary occlusion and reperfusion isdescribed in McNulty et al., J. Am. Physiol., 1996, H2283-9.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise that as specifically describedherein.

1-103. (canceled)
 104. A method of detecting, imaging or monitoringcongestive heart failure in a patient, comprising the steps of:administering a diagnostic agent to the patient; and acquiring an imageof a site of concentration of the diagnostic agent in the patient by adiagnostic imaging technique; wherein the diagnostic agent comprises amatrix metalloproteinase inhibitor and an imaging moiety conjugated tosaid matrix metalloproteinase inhibitor; and wherein said imaging moietyis a gamma ray radioisotope, a positron emitting radioisotope, amagnetic resonance imaging contrast agent, an x-ray contrast agent, oran ultrasound contrast agent.
 105. A method of detecting, imaging ormonitoring congestive heart failure in a patient according to claim 104,wherein the diagnostic agent comprises an imaging moiety and a compoundof the formula:(Q)_(d)-(L_(n))_(x″)-K or a pharmaceutically acceptable salt thereof;wherein Q is a matrix metalloproteinase inhibitor; L_(n) is an optionallinking group; K is a chelator or a surfactant capable of forming anechogenic gas filled lipid sphere or microbubble; d is 1, 2, 3, 4, 5, 6,7, 8, 9, or 10; and x″ is 0 or
 1. 106. A method of detecting, imaging ormonitoring congestive heart failure in a patient according to claim 105,wherein d is 1, 2, 3, 4, or
 5. 107. A method of detecting, imaging ormonitoring congestive heart failure in a patient according to claim 105,wherein d is 1, 2, or
 3. 108. A method of detecting, imaging ormonitoring congestive heart failure in a patient according to claim 105,wherein d is 1 or
 2. 109. A method of detecting, imaging or monitoringcongestive heart failure in a patient according to claim 105, wherein dis
 1. 110. A method of detecting, imaging or monitoring congestive heartfailure in a patient according to claim 105, wherein x″ is
 0. 111. Amethod of detecting, imaging or monitoring congestive heart failure in apatient according to claim 105, wherein x″ is
 1. 112. A method accordingto claim 104, wherein said imaging moiety is a paramagnetic metal, aferromagnetic metal, a gamma-emitting radioisotope, positron-emittingradioisotope, or an x-ray absorber.
 113. A method according to claim104, wherein said imaging moiety is ^(99m)Tc, ⁹⁵Tc, ¹¹¹In, ⁶²Cu, ⁶⁴Cu,⁶⁷Ga, or ⁶⁸Ga.
 114. A method according to claim 104, wherein saidimaging moiety is ⁹⁹ ^(m) Tc.
 115. A method according to claim 104,wherein said imaging moiety is ¹¹¹In.
 116. A method according to claim104, wherein said imaging moiety is Gd(III), Dy(III), Fe(III), orMn(II).
 117. A method according to claim 104, wherein said imagingmoiety is Re, Sm, Ho, Lu, Pm, Y, Bi, Pd, Gd, La, Au, Au, Yb, Dy, Cu, Rh,Ag, and Ir.
 118. A method according to claim 104, wherein said imagingmoiety is a perfluorocarbon gas or sulfur hexafluoride.
 119. A methodaccording to claim 118, wherein said perfluorocarbon gas isperfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane,perfluorocyclobutane, perfluoropentane, or perfluorohexane.